Plant Biotechnology Vol.32 No.1


Original Papers

α-Amylase from Mon Thong durian (Durio zibethinus Murr. cv. Mon Thong)-nucleotide sequence analysis, cloning and expression

Saijai Posoongnoen, Raksmont Ubonbal, Sompong Tammasirirak, Jureerut Daduang, Hiromichi Minami, Kenji Yamamoto, Sakda Daduang . . . . . . . 1 [PDF]

Amylase is hypothesized to involve in the initiation of intracellular starch granule digestion in the plastids of ripening durians. A putative α-amylase from Mon Thong durian (Durio zibethinus Murr. cv. Mon Thong; DzAmy3) was successfully isolated and its gene contain a 2,679 base pair open reading frame, which encodes 892 amino acids with a calculated molecular mass of 93.7 kDa and an isoelectric point of 5.77. The DzAmy3 contains starch binding domain with a putative plastid transit peptide and α-amylase like domain. Phylogenetic tree analysis proved it into the family three of plant α-amylases. The predicted structural model proposed a catalytic triad (Asp611, Glu636 and Asp719) for general acid/base hydrolysis. Recombinant DzAmy3 (rDzAmy3) was successfully expressed in Escherichia coli. rDzAmy3 hydrolyzed starch and ethylidene-pNP-G7 which confirms the authenticity of DzAmy3 gene and functional α-amylase. The rDzAmy3 had an optimum activity at pH 8.0 and 30℃. It was stable in the pH range of 7–8 at 37℃, temperature range of 5–20℃ and in the presence of 1% (v/v) Tween 20 and Triton X-100. Substrate specificity analysis revealed that rDzAmy3 was active toward β-limit dextrin, starch, amylopectin, amylose and glycogen.

The Arabidopsis ubiquitin ligase ATL31 is transcriptionally controlled by WRKY33 transcription factor in response to pathogen attack

Thais Huarancca Reyes, Shugo Maekawa, Takeo Sato, Junji Yamaguchi . . . . . . . 11 [PDF]

ATL31, an Arabidopsis RING-type ubiquitin ligase, plays a critical role in plant carbon/nitrogen (C/N)-nutrient responses during post-germinative growth and in defense responses to pathogen attack. ATL31 expression under these stress conditions suggested the presence of transcriptional regulators mediated by these stress signals. We recently reported that the expression pattern of WRKY33, a transcription factor involved in plant defense responses, is highly correlated with that of ATL31. In this study, we investigated the detailed relationship between the ATL31 gene and WRKY33. Using transient reporter analysis, we found that WRKY33 could significantly activate ATL31 transcription in plant cells. Transcript analysis of stable transgenic Arabidopsis plants overexpressing WRKY33 confirmed that the expression of ATL31 in response to the PAMPs flg22 and chitin was enhanced compared with wild-type plants, while expression was repressed in wrky33 mutants.
Further detailed transient reporter analysis revealed that transactivation by WRKY33 is required and mediated through a specific W-box cis-acting element in the promoter region of the ATL31 gene. In contrast, WRKY33 did not regulate ATL31expression during the C/N response. Taken together, these results demonstrate that WRKY33 acts as a transcription factor of ATL31 and positively regulates its expression during activation of plant defense responses.

Continuous expression of genes for xylem cysteine peptidases in long-lived ray parenchyma cells in Populus

Satoshi Nakaba, Naoki Takata, Makoto Yoshida, Ryo Funada . . . . . . . 21 [PDF]

XYLEM CYSTEINE PEPTIDASE 1 (XCP1) and XCP2 are key autolytic enzymes in programmed cell death of short-lived tracheary elements in plants. However, the patterns of expression of XCP genes remain to be clarified in long-lived ray parenchyma cells, which survive for several years after the completion of secondary wall formation. We isolated full-length cDNAs that encoded three XCPs (PsgXCP1, PsgXCP2A and PsgXCP2B) of Populus. The deduced amino acid sequences of the XCPs revealed three conserved catalytic residues, namely, cysteine (Cys), histidine (His) and asparagine (Asn), indicating that PsgXCP1, PsgXCP2A and PsgXCP2B might function as papain-like cysteine proteases. Ray parenchyma cells in hybrid poplar remained alive for several years, with continuous expression of XCP genes even after obvious secondary wall thickenings. Levels of expression of XCP genes were lower in the innermost annual ring of sapwood than in one and/or two outer annual rings from beyond the innermost sapwood. This result implies that autolysis of ray parenchyma cells might not be fully explained by dramatically enhanced expression of XCP genes just before their programmed death. In addition, the expression of PtaSND1-A1, PtaVND6-A1 and PtaMYB003 genes was detected in all annual ring in sapwood. Our observations suggest that the expression of genes associated with xylem differentiation such as secondary wall formation and cell death continues for several years in long-lived ray parenchyma cells before their death.

Live-imaging evaluation of the efficacy of elevated CO2 concentration in a closed cultivation system for the improvement of bioproduction in tomato fruits

Haruaki Yamazaki, Nobuo Suzui, Yong-Gen Yin, Naoki Kawachi, Satomi Ishii, Hiroaki Shimada, Shu Fujimaki . . . . . . . 31 [PDF]

To maximize fruit yield of tomatoes cultivated in a controlled, closed system such as a greenhouse or a plant factory at a limited cost, it is important to raise the translocation rate of fixed carbon to fruits by tuning the cultivation conditions. Elevation of atmospheric CO2 concentration is a good candidate; however, it is technically difficult to evaluate the effect on fruit growth by comparing different individuals in different CO2 conditions because of large inter-individual variations. In this study, we employed a positron-emitting tracer imaging system (PETIS), which is a live-imaging technology for plant studies, and a short-lived radioisotope 11C to quantitatively analyze immediate responses of carbon fixation and translocation in tomatoes in elevated CO2 conditions. We also developed a closed cultivation system to feed a test plant with CO2 at concentrations of 400, 1,500 and 3,000 ppm and a pulse of 11CO2. As a result, we obtained serial images of 11C fixation by leaves and subsequent translocation into fruits. Carbon fixation was enhanced steadily by increasing the CO2 concentration, but the amount translocated into fruits saturated at 1,500 ppm on average. The translocation rate had larger inter-individual variation and showed less consistent responses to external CO2 conditions compared with carbon fixation. Our experimental system was demonstrated to be a valuable tool for the optimization of closed cultivation systems because it can trace the responses of carbon translocation in each individual, which are otherwise usually masked by interindividual variation.

Double-stranded DNA introduction into intact plants using peptide–DNA complexes

Manoj Lakshmanan, Takeshi Yoshizumi, Kumar Sudesh, Yutaka Kodama, Keiji Numata . . . . . . . 39 [PDF]

Introducing exogenous genes into plant cells is an essential technique in many fields in plant science and biotechnology. Despite their huge advantages, disadvantages of current transfection methods include the requirement of expensive equipment risk of gene damage, low transformation efficiency, transgene size limitations, and limitations of applicable plant types. Recently developed peptide-based gene carriers can deliver plasmid and double-stranded RNA. However, the delivery of double-stranded DNA (dsDNA), specifically PCR products, has not been studied. As dsDNA is handled in several plant science labs, peptide-based gene carriers are expected to be applicable to dsDNA in addition to plasmid DNA and double-stranded RNA. Here, we demonstrate dsDNA introduction into intact Nicotiana benthamiana leaves by using an ionic complex of a fusion peptide comprising (KH)9 and Bp100 with dsDNA encoding Renilla luciferase as a reporter gene. The buffer condition for the complex preparation and infiltration significantly affected the transfection efficiency; this is because the structure of the complex in various protonated conditions contributed to the transfection efficiency. Structures of the complex and peptide are key factors for improving the peptide-based gene delivery system for plants.

High-level expression of sucrose inducible sweet potato sporamin gene promoter: β-glucuronidase fusion gene in transgenic Nicotiana plumbaginifolia

Youhei Honma, Takashi Yamakawa . . . . . . . 47 [PDF]

We produced transgenic Nicotiana plumbaginifolia plants which contained Spomin (sporamin minimal promoter)-GUS fused chimeric gene constructs with 5 types of signal sequences, such as cytosol, apoplast, ER, vacuole and plastid, and analyzed the GUS expression patterns after sucrose treatment. Spomin induced extremely high GUS activities after 6% and 10% sucrose treatment, especially in leaves. The high GUS activities were observed in leaves of the Spomin-ER-GUS construct treated with 6% or 10% sucrose. These were over 200 times higher than those in leaves with the 35S promoter-ER-GUS construct. The 10% sucrose treatment significantly altered GUS activities in all Spomin and 35S promoter constructs compared with those in the 6% sucrose treatment; some increased and some decreased. GUS activities in 2 months old plants were almost the same as 8 month old plants, indicating that GUS expression driven by Spomin was stably maintained. Also, even when sucrose treatment was stopped, GUS gene expression by Spomin continued for 10 days.

Development of a new high-throughput method to determine the composition of ten monosaccharides including 4-O-methyl glucuronic acid from plant cell walls using ultra-performance liquid chromatography

Shingo Sakamoto, Kouki Yoshida, Satoko Sugihara, Nobutaka Mitsuda . . . . . . . 55 [PDF]

Plant cell walls are an important dietary source for livestock, and could be an enormous resource for production of next-generation bioethanol and more valuable materials. Because polysaccharides are major components of plant cell walls, analysis of their composition is important. In this report, we established a high-throughput method to determine the composition of ten monosaccharides from plant cell walls simultaneously using ultra-performance liquid chromatography with p-aminobenzoic ethyl ester-labeling technology. Complete separation of a mixture of internal standards, 2-deoxyglucose and 3-O-methyl glucose, and ten monosaccharides, consisting of seven neutral and three acidic sugars including 4-O-methyl-D-glucuronic acid, which are frequently found in plant cell wall polysaccharides, can be obtained within 7 min using this system. Relative standard deviations of retention time and peak area value are lower than 1%. Linearity for broader dynamic ranges (0.02–2000 mg l−1), faster analysis and higher sensitivity than other traditional methods, including one that employs widely used high-performance anion exchange chromatography, are achieved. We evaluated this new method by analyzing the composition of cell walls from three model plants (Arabidopsis thaliana, rice and hybrid aspen) and confirmed that the obtained results for most monosaccharides are consistent with those in previous studies. These data suggest that our newly developed system could greatly contribute to the study of plant cell walls, especially research requiring highthroughput analysis.

Altered levels of primary metabolites in response to exogenous indole-3-acetic acid in wild type and auxin signaling mutants of Arabidopsis thaliana: A capillary electrophoresis-mass spectrometry analysis

Aya Anegawa, Miwa Ohnishi, Daisuke Takagi, Chikahiro Miyake, Chizuko Shichijo, Kimitsune Ishizaki, Hidehiro Fukaki, Tetsuro Mimura . . . . . . . 65 [PDF]

A comprehensive analysis of the levels of primary metabolites in wild type (WT) and several auxin-signaling mutants namely, tir1, slr and arf7 arf19 of Arabidopsis thaliana has been performed using CE-MS, a technique particularly sensitive for the measurement of polar compounds. We first measured the levels of primary metabolites in shoots and roots, most of the analyzed metabolites were found to be quantitatively and qualitatively comparable in WT and three kinds of mutants (tir1, slr and arf7 arf19). Some amino acids such as GABA, Arg, Orn, Val, Thr, Leu and Ile exhibited a unique pattern of distribution between shoots and roots in both WT and the mutants. On the other hand, the mutant slr showed a quite different pattern of metabolites measured in the present study.  Subsequently, the responses of primary metabolites to a short-term (60 min) application of exogenous IAA (10−7, 10−8 M) in WT and the mutants were characterized. Due to IAA treatments, some amino acids such as GABA in WT roots and Gly and Ala in WT shoots were altered, but not in the mutants. Gln was altered in slr shoots by 10−7 M IAA treatment. Levels of G6P from the glycolic pathway were altered in WT roots and those of 2PG, 3PG were altered in tir1 shoots in response to IAA treatments. The levels of succinate in TCA cycle were altered by IAA treatments in WT shoots but not in the mutants. IAA treatment inhibited the respiration in WT roots. The suppression of respiration might account for the IAA-dependent alteration of some metabolites. Difference of auxin responses between WT and auxin-signaling mutants suggests that some metabolic processes are under IAA control.

In planta comparative analysis of improved green fluorescent proteins with reference to fluorescence intensity and bimolecular fluorescence complementation ability

Yuta Fujii, Yutaka Kodama. . . . . . . . 81 [PDF]

Green fluorescent protein (GFP) was discovered from the jellyfish Aequorea victoria, and several improvements have been carried out to change its physicochemical properties. The resulting improved GFP variants have been used as reporter proteins for bioimaging techniques in various research fields including plant science. Almost all GFP variants were developed using Escherichia coli to improve fluorescence properties in mammalian cells, but the impact in other organisms such as plant cells remains to be determined. In this study, we performed comparative analysis of four improved GFP variants, GFP-S65T, eGFP, frGFP and sfGFP, with reference to the fluorescence intensity in Arabidopsis protoplasts, and found that sfGFP is the brightest. Using non-fluorescent fragments from the GFP variants, we also conducted bimolecular fluorescence complementation (BiFC) assays to find appropriate fragment pairs of GFP-based BiFC for visualization of protein–protein interactions in living plant cells. Our observations revealed that the brightest is the sfGFP-based BiFC. Further, as an evaluation method for the sfGFP-based BiFC, a BiFC competition assay was successfully completed for the first time in planta. The present study provides useful information for selection and improvement of the GFP molecule and its application to BiFC technology in plants.

Polycistronic expression of RNA silencing suppressor protects its own mRNA from RNA silencing

Yukio Kurihara, Emiko Okubo-Kurihara, Minami Matsui . . . . . . . 89 [PDF]

The internal ribosome entry site (IRES) enables polycistronic expression of multiple genes from a single mRNA controlled by one promoter. In general, only the most 5′ coding sequence is abundantly translated from polycistronic mRNAs in eukaryotic cells; IRES-mediated translation allows additional coding sequences at other positions to be translated at detectable levels. However, IRES-mediated translation often results in much lower protein expression than translation of single-gene mRNAs. We first aimed to improve IRES-mediated gene expression with a transient expression system based on Nicotiana benthamiana. We demonstrated that the presence of two cassettes comprised of an IRES-mediated Venus coding sequence results in higher Venus expression than one cassette. The double IRES cassette system is expected to be useful for expressing a gene of interest polycistronically in plants. Using the double IRES cassette system, we found that polycistronic expression of a reporter gene and two copies of an IRES-mediated RNA silencing suppressor gene protected the transcribed mRNA from RNA silencing-mediated degradation. This is the first report of a mRNA self-protection system from RNA silencing by IRES-mediated expression of a viral suppressor in plants.


The shoot of Ranunculus nipponicus var. submersus, a submerged vascular plant, can actively take up nitrate from cool water

Shu Takayanagi, Yuma Takagi, Hiroshi Hasegawa . . . . . . . 97 [PDF]

Nitrate uptake characteristics in the shoots of Ranunculus nipponicus var. submersus (Japanese name: Baikamo),a submerged eudicot adapted to groundwater temperature (approximately 15℃), were investigated for the phytoremediationof nitrate-polluted groundwater. For the experiments, we developed a culture system that allows R. nipponicus growth under laboratory conditions. 15N-nitrate feeding experiments showed that apparent nitrate uptake by the shoots of R. nipponicus was approximately three times of that by the shoots of Egeria densa, a model submerged monocot, at 15℃. A DNA fragment of the R. nipponicus high-affinity nitrate transporter (NRT2) was isolated and the deduced amino acid sequence of the partial RnNRT2 protein was similar to that of NRT2 in monocots rather than in eudicots. Real-time reverse transcription- PCR analysis revealed that after the shoots were fed 0.2 mM nitrate, RnNRT2 transcripts in the shoots of R. nipponicus were induced within 1 h, reached a maximum by 6 h and then decreased. At 15℃, RnNRT2 transcripts in the shoots of R. nipponicus, in contrast to EdNRT2 transcripts in shoots of E. densa, were rapidly and strongly induced by nitrate. We concluded that the shoots of R. nipponicus have a system of high-affinity nitrate uptake actively functioning under cool conditions (15℃) and may be useful for the clean-up of nitrate-contaminated groundwater.

Arabidopsis LAZY1 is a peripheral membrane protein of which the carboxy-terminal fragment potentially interacts with microtubules

Shu Sasaki, Kotaro T. Yamamoto . . . . . . . 103 [PDF]

LAZY1 is a protein involved in gravity signaling of shoot gravitropism of rice, maize and Arabidopsis. Although the lazy1 mutants have been well-characterized, the function of the LAZY1 protein is still largely unknown. In this study, we used fluorescence microscopy to examine the subcellular localization of Arabidopsis LAZY1 (AtLAZY1) and its truncated proteins fused to GFP in tobacco leaves. We found that AtLAZY1 localizes to the plasma membrane through the C-terminal region, suggesting that the putative trans-membrane domain in the N-terminal half is not required for localization. Next, we took a biochemical approach to investigate the membrane association of AtLAZY1. Transiently expressed AtLAZY1 in transgenic Arabidopsis was fractionated in an insoluble fraction that contained membranous compartments. AtLAZY1 was solubilized by a non-ionic detergent or at a high pH condition, suggesting that AtLAZY1 is a peripheral membrane protein. We also found that when expressed in tobacco the C-terminal part of AtLAZY1 co-localized with microtubules. A microtubule binding assay showed that the C-terminal half of AtLAZY1, which localized to the plasma membrane, interacted with microtubules in vitro. These results suggest that AtLAZY1 may function with microtubules at the periphery of the plasma membrane in the gravity signaling process.