Plant Biotechnology Vol.31 No.3


Original Papers

High speed and high precision optical interferometric technique to investigate instantaneous growth related changes of plant leaves

Bodhipaksha Lalith Sanjaya Thilakarathne, Uma Maheswari Rajagopalan, Hirofumi Kadono, Tetsushi Yonekura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 [PDF]

Instantaneous growth related changes of leaves of Radish, Soybean and rice plants were investigated with a highspeed and ultra-high accuracy optical interferometer. The interferometer, called statistical interferometer, SIT (Statistical Interferometric Technique) is a real-time, non-contact, and robust one and has a subnanometric accuracy and can measure leaf elongation changes every 500 ms. SIT revealed the existence of nanometric fluctuations in the elongation of leaves. Control experiments with dead and mature leaves did indeed establish the existence of null or smaller standard deviations in the fluctuations in contrast to the large deviations obtained for young and healthier leaves. Moreover, the standard deviations of the fluctuations were found to be sensitive to environment of ozone(O3), a secondary atmospheric pollutant. Long term O3 exposure with Radish and Soybean leaves led to a decrease in the standard deviations of nanometric fluctuations with increase in O3 concentrations which correlated with a decrease of gas exchange measures. Under short term O3 exposures for 3 h with two Japanese rice cultivars, cv. Koshihikari and cv. Fusaotome that are known to have varying O3 tolerance made the nanometric fluctuations to decrease in comparison to gas exchange measures which showed no variation. The fluctuations could also differentiate the cultivar’s sensitivity to O3 with cv. Koshihikari showing a drastic reduction in comparison to cv. Fusaotome. The results suggest that the nanometric fluctuations may possibly relate to the life maintaining physiological state of the plant and thus may act as an indicator of the vitality of the plant.

Effect of nitrate concentration in nutrient solution on hemagglutinin content of Nicotiana benthamianaleaves in a viral vector-mediated transient gene expression system

Naomichi Fujiuchi, Ryo Matsuda, Nobuyuki Matoba, Kazuhiro Fujiwara. . . . . . . . . . . . . . . . . . . . . . . . . . . 207 [PDF]

Viral vector-mediated transient expression enables rapid biopharmaceutical protein production using whole plants. In recent years, plant growth conditions have received close attention as factors influencing the content of recombinant protein. In particular, because nitrogen is an important resource used in protein synthesis, nitrogen supplied to the root zone may influence accumulation of recombinant proteins. We tested whether fertilization of Nicotiana benthamiana with high-nitrate nutrient solution during plant growth prior to Agrobacterium-mediated delivery (agroinfiltration) of a tobamoviral vector resulted in high recombinant influenza hemagglutinin (HA) content per unit of leaf fresh weight and/or per plant. Nutrient solution containing 60 mM nitrate yielded 40% higher HA content per unit of leaf fresh weight, and comparable HA content per plant, relative to nutrient solution containing 12 or 36 mM nitrate. There were positive correlations among HA, total soluble protein (TSP), and soluble reduced-nitrogen content, suggesting that fertilization with the high-nitrate nutrient solution increased soluble reduced nitrogen and subsequently provided high TSP and HA. Although the increased HA content per unit of leaf fresh weight was offset by reduced leaf fresh weight on a per plant basis, nitrate-enriched fertilization represents an easily applicable technique to obtain a given amount of HA from smaller leaf biomass, thereby potentially reducing downstream processing costs for HA vaccine production using viral vector-mediated transient expression systems.

Functional and molecular characteristics of rice and barley NIP aquaporins transporting water, hydrogen peroxide and arsenite

Maki Katsuhara, Shizuka Sasano, Tomoaki Horie, Tadashi Matsumoto, Jiye Rhee, Mineo Shibasaka. . . . 213 [PDF]

Mercury-sensitive water transport activities were detected in seven NIP (Nodulin 26-like intrinsic protein) type aquaporins among eleven NIPs examined. Amino acid substitutions in rice OsNIP3;3 revealed that mercury-sensitivity depended on a histidine (but not on a cysteine) in apoplastic loop C in plant NIP aquaporins, although the cysteine is involved in the mercury-sensitivity of animal aquaporins. Rice OsNIP3;3 was also first identified as a unique aquaporin facilitating all water, hydrogen peroxide and arsenite transports. In rice OsNIP3;2, hydrogen peroxide and arsenite transport activities were detected, but water transport was not. Barley HvNIP1;2- or rice OsNIP2;1-expressing yeast cells showed the arsenite transport activity but not the H2O2 transport activity. The present work revealed novel molecular mechanisms of water and other low molecular weight compounds transport/selection in barley and rice NIP aquaporins, including the histidine-related mercury-sensitivity in the water transport of aquaporins.

Enhanced translation of the downstream ORF attributed to a long 5' untranslated region in the OsMac1 gene family members, OsMac2 and OsMac3

Hiromi Aoki, Hiroshi Teramura, Mikhail Schepetilnikov, Lyubov A Ryabova, Hiroaki Kusano, Hiroaki Shimada . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 [PDF]

The 5′-untranslated region (5′UTR) of mRNAs often affects the translational efficiency of the downstream open reading frames (ORFs), and some of its regulatory elements are involved in the initiation of translation. We found that the 5′UTR of the rice OsMac1 mRNA, which consisted of more than 500 nucleotides, yielded a significant enhancement of the translational efficiency of the downstream ORF. In the rice genome, OsMac1 represents a conserved gene family with two homologues, OsMac2 and OsMac3, which contain DUF300 (domain of unknown function 300) domains with predicted transmembrane regions. Similarly to the OsMac1 mRNA, the OsMac2 and OsMac3 mRNAs possess long 5′UTRs consisting of 312 and 318 nucleotides, respectively, that precede the main ORFs, which allow the elevation of the translational efficiency of the downstream ORF. The estimation of the translational efficiency of the GUS gene, which is located after the 5′UTRs, in suspension cultures of rice protoplasts showed that it was significantly greater than that of the control. These results suggest that 5′UTRs of OsMac2 and OsMac3 enhance the translation of the downstream ORF. Our results indicate that these 5′UTRs play a role of novel translational enhancer elements that enable the efficient translation of the downstream ORF.

Regulation of xanthophyll cycle pool size in response to high light irradiance in Arabidopsis

Yuiko Kawabata, Satomi Takeda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 [PDF]

The xanthophyll cycle is known to play a key photoprotective role in plants. While it has been demonstrated that the pool size of xanthophyll cycle pigments (VAZ) increases during acclimation to high light (HL) intensity, the associated regulatory mechanism remains largely unknown. Since the redox state of Plastoquinone (PQ) is thought to influence the expression of photosynthesis-related genes, we addressed the possibility that the redox state of PQ affects the regulation of carotenoid synthesis. To do this, we used a photoautotrophic cell culture of Arabidopsis, that can grow stably in sugar-free medium, and regulated the redox state by adding the electron transport inhibitors, DCMU or 2,5-dibromo-3-methyl-6- isopropyl benzoquinone (DBMIB) to the cells. We then analyzed their effects on VAZ accumulation and the expression of genes encoding carotenoid biosynthesis enzymes. The VAZ pool size and the transcript levels of β-carotene hydroxylase genes (Chy1, Chy2, CYP97A3 [Lut5]) were higher under HL than under normal conditions; however, DCMU treatment partially blocked these effects. In contrast, DBMIB treatment increased VAZ accumulation and transcription of these genes without HL irradiation to a certain extent. Based on these results, we propose that the redox state of PQ is one of the regulator of the pool size of VAZ. The expression of LcyB encoding lycopene β-cyclase was also clearly up-regulated by HL, but not affected by PQ redox. In addition, using mutants of the β-carotene hydroxylase genes, we identified Chy1 as the gene that contributed most to the increase in VAZ pool size by HL.

A watermarking system for labeling genomic DNA

Naoki Yamamoto, Hiroyuki Kajiura, Shinya Takeno, Nobuaki Suzuki, Yoshihisa Nakazawa . . . . . . . . . . . 241 [PDF]

We established a DNA watermarking system for discriminating transgenic plants. The system contains an encryption algorithm based on a binary system, genetic transformation and a detection algorithm for encrypted DNA watermark sequences using a DNA dot plot. The encryption algorithm converted character strings into nucleic acid sequences through binary digits, and the sequence was designed to be resistant to transition mutations to decipher codes completely. Moreover, the encrypted sequences were capable of taking specific nucleotide sequences in using the algorithmic redundancy of the corresponding DNA. Genetic transformation enables labeling plant genomes with DNA watermarks. The detection algorithm allows finding traces of sequence changes in DNA watermarks, complementing the error protection function of the encryption algorithm. To validate the effectiveness of our DNA watermarking system, we introduced a DNA watermark to the tobacco genome and detected the DNA watermark in PCR products amplified from the genome. This indicates that DNA watermark technology is useful for introducing artificial genetic markers in plant organisms, in particular when several transgenic host plants and transgenes are used. The source codes of the Perl scripts are available in this report.

Micropropagation of Stevia rebaudiana Betroni and assessment of genetic stability of in vitro regenerated plants using inter simple sequence repeat (ISSR) marker

Hemaid Ibrahim Ahemaidan Soliman, Ehab Mohamed Rabei Metwali, Omar Abdel-Hakeim Almaghrabi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 [PDF]

Stevia rebaudiana Bertoni has become a new sweetener crop in different regions in the world. Stevia produces few seeds, therefore, micropropagation is a prevalent method to obtain sufficient amount of uniform plants. In the present study, in vitro propagation of Stevia was attempted through multiple shoot regeneration from nodal segments cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyladenine (BA), α-napthyalene acetic acid (NAA), indole acetic acids (IAA) and thidiazuran (TDZ). The maximum of number axillary shoots per explant (3.24) and highest shoot length (3.12 cm) were observed with MS medium supplemented with 1.0 mg l−1 BA+0.05 mg l−1 NAA and 2.0 mg l−1 BA+0.05 mg l−1 NAA, respectively. Roots were produced within two weeks and the highest percentage (98.72%) of root induction, the maximum number of roots (9.46 roots/shoot) and root length (9.87 cm) were recorded on MS medium fortified with 0.5 mg l−1 IAA and 1.5 mg l−1 IAA, respectively. The ex-vitro plantlets were successfully acclimatized with a survival rate of 70% at the hardening phase. Inter simple sequence repeat (ISSR) markers were used to analyze the genetic stability of micro-propagated and mother plants of Stevia. Four ISSR primers generated clear, distinct and reproducible bands. All ISSR profiles from micro-propagated plants were monomorphic and similar to mother plants, while low variation was induced in the next sub-culture. The results indicated that Stevia plantlets regenerated using micropropagation techniques standardized at our lab were genetically stable especially in the early sub-cultures.

Substrate-enantiomer selectivity of matairesinol O-methyltransferases

Safendrri Komara Ragamustari, Masaomi Yamamura, Eiichiro Ono, Takefumi Hattori, Shiro Suzuki, Hideyuki Suzuki, Daisuke Shibata, Toshiaki Umezawa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 [PDF]

Previously we reported a cDNA encoding an O-methyltransferase (OMT) responsible for the O-methylation of matairesinol to afford arctigenin in Carthamus tinctorius. However, the regioselectivity and stereoselectivity of its reaction are not yet well understood. In this paper, we report the characterization and comparison of three matairesinol OMTs (MROMTs) encoded by cDNAs isolated from C. tinctorius (CtMROMT), Anthriscus sylvestris (AsMROMT), and Forsythia koreana (FkMROMT). Although they shared matairesinol as a substrate, each recombinant MROMT showed different catalytic behavior. AsMROMT and CtMROMT methylated matairesinol’s hydroxyl group at the C4′ position giving rise to arctigenin, while FkMROMT methylated the C4 position hydroxyl group giving rise to isoarctigenin. Analysis of the enantiomeric composition of products from racemic matairesinol showed all OMT reactions to be highly selective in terms of the substrate enantiomers and only use the (−)-enantiomer as the substrate.


ShiftedIonsFinder: A standalone Java tool for finding peaks with specied mass differences by comparing mass spectra of isotope-labeled and unlabeled data sets

Kota Kera, Yoshiyuki Ogata, Takeshi Ara, Yoshiki Nagashima, Norimoto Shimada, Nozomu Sakurai, Daisuke Shibata, Hideyuki Suzuki . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 [PDF]

In mass spectrometry (MS)-based metabolite annotation, the prediction of theoretical elemental compositions from accurate mass is effective to support database search results or annotation for unknown metabolites. However, there are deviations (error) from the accurate mass such that accurate estimation of elemental composition may not be possible. A technique to label the biological sample with a stable isotope is effective to solve this problem, but the software for handling labeled MS data is limited. In this paper, we present a standalone Java graphical user interface (GUI) program tool—ShiftedIonsFinder—that helps find peaks having specified mass differences by comparing the mass spectra between two data sets. Using this tool, it is possible to select candidate labeled peaks by comparing a sample labeled with a stable isotope against an unlabeled sample. This tool also detects peaks with chemical modifications, such as glycosylation and acylation. Since the search results can be generated in Excel format, the user can easily perform further analyses or edit the list as needed. ShiftedIonsFinder ( is available free of charge at KOMICS.

Influence of phenyl-urea and adenine-type cytokinins on direct adventitious shoot regeneration of cabbage (Brassica oleracea subsp. capitata) “KY Cross”

Seyed Ali Ravanfar, Shahida Salim, Maheran Abd Aziz, Siti Nor Akmar Abdullah, Azmi Abdul Rashid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 [PDF]

In this study, the effects of phenyl-urea (Thidiazuron) and adenine (6-benzylaminopurine) and 6- y,y,dimethylally-amino purine type cytokinins alone or in combination with indole-3-butyric acid on shoot regeneration from hypocotyl and cotyledonary explants of Brassica oleracea ssp. capitata “KY Cross” were investigated. For hypocotyl explants, medium containing 2.27 μM Thidiazuron showed the highest mean number (18.15) of separable shoots per explant with 80% shoot formation. In the case of cotyledonary explants, the highest mean number of shoots (3.03) was obtained on medium containing 12.30 μM 6-y,y,dimethylally-amino purine with a percentage of 56.67% shoot formation. Plantlets were successfully acclimatized with 70% survival in potting medium consisting of coconut husk+vermicompost (7 : 1 v/v). The regeneration system developed herewith will be a valuable tool for genetic improvement of cabbage “KY Cross”.

Cryopreservation of mat rush lateral buds by air dehydration using aluminum cryo-plate

Takao Niino, Wunna, Kazuo Watanabe, Naoko Nohara, Tariq Raque, Shin-ichi Yamamoto, Kuniaki Fukui, Miriam Valle Arizaga, Carlos Roman Castillo Martinez, Toshikazu Matsumoto, Florent Engelmann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 [PDF]

A cryopreservation protocol based on air dehydration of explants placed on aluminium cryo-plates, termed D cryo-plate, was successfully developed for in vitro mat rush (Juncus decipiens Nakai) lateral buds. The buds of line ‘Hiroshima 4gou(1)’ with basal stems were dissected from multiple shoots and precultured overnight at 25°C on solid MS medium containing 0.3 M sucrose. Precultured buds were placed on aluminium cryo-plates, each one with 10 elliptical wells (2.5 mm long, 1.5 mm wide and 0.75 mm deep) and embedded in calcium alginate gel with 0.4 M sucrose and 1 M glycerol. Osmoprotection was performed by immersing the cryo-plates with buds for 30 min at 25°C in loading solution (2 M glycerol+1.0 M sucrose). Buds were dehydrated to 26% moisture content (fresh weight) by placing the cryo-plates for 2.5 h either in the air current of a laminar flow cabinet or in Petri dishes containing silica gel. Cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, cryo-plates were immersed in liquid medium with 1.0 M sucrose for 15 min at room temperature. Under these conditions, regrowth of cryopreserved buds of line, ‘Hiroshima 4gou (1)’ was 93% after four weeks culture. The average regrowth of 20 mat rush lines was 88%. The D cryo-plate procedure will facilitate cryostorage of mat rush germplasm.

Identification of proteins that interact with a plant nuclear protein using the yeast split-Trp sensor

Chikako Kubo, Yoshihiro Nomura, Naoki Matsuo, Hiroharu Banno . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 [PDF]

The original yeast two-hybrid system is based on the functional reconstitution of the transcriptional activator of a reporter gene by means of the interaction between bait and prey proteins, which possess DNA-binding and transactivation domains, respectively. However, in certain cases, interaction between a fusion protein with a DNA-binding domain and a bait peptide can activate the transcription of a reporter gene, irrespective of the interaction between the bait and prey proteins. Therefore, this system cannot be used to screen for prey proteins that specifically interact with the “bait” because bait peptides initiate transactivation. We used a split-Trp sensor to identify protein interactions with the C-terminal region of Arabidopsis Enhancer of Shoot Regeneration 1(ESR1), which has transactivating capabilities in yeast cells. ESR1- Interacting Candidate 1(EIC1) was identified using this system, and although its functions are unknown, EIC1 was observed to localize to nuclei when its GFP-fusion protein was expressed in onion epidermal cells. These results suggest that the split- Trp sensor could be useful in screening for proteins that interact with peptides that have transactivation domains in yeast cells.