Plant Biotechnology Vol.31 No.2

Contents

Original Papers

Transgenic tobacco and tomato plants expressing Wasabi defensin genes driven by root-specic LjNRT2 and AtNRT2.1 promoters confer resistance against Fusarium oxysporum

Kynet Kong, Valentine Otang Ntui, So Makabe, Raham Sher Khan, Masahiro Mii, Ikuo Nakamura. . . . . . . 89 [PDF]

Antifungal peptides are a potential group of defense molecules that have been utilized to develop resistance to various plant pathogens. Wasabi defensin (WD) gene (0.5 kb) consists of cysteine-rich peptides that show potent growth inhibition of pathogenic filamentous fungi, such as Botrytis cinerea. Under regulation by the root-specific LjNRT2 or AtNRT2.1 promoter, WD gene was expressed in the roots of transgenic tobacco and tomato plants by Agrobacterium-mediated transformation. The regenerated plants showed stable integration of the transgene, with different insertion sites, and the transgene was expressed in the root tissues but not in the leaf tissues. This result confirmed that WD protein accumulated only in the roots of transgenic plants. In a bioassay for resistance to Fusarium oxysporum, all transgenic plants showed increased resistance to the fungus as compared to non-transformed plants. Protein extracts from root and leaf tissues were assayed for antifungal activity and the activity was express as the number of colonies formed per cm2 (CFU cm−2). The CFU values of the root and leaf extracts of control plants did not show significant differences. In contrast, the CFU values of the root extracts of the transgenic plants were significantly lower than those of the leaf extracts and much lower than those of control. These results suggest that LjNRT2 and AtNRT2.1 promoters triggered the antifungal gene expression in the roots and conferred increased resistance to the root pathogen F. oxysporum. In the view of bio-safety, the root-specific expression of the transgene is desirable because the roots of tomato are not edible.

High concentrations of sucrose induce overwintering bud formation in gentian plantlets cultured in vitro

Tomohiro Imamura, Atsumi Higuchi, Ken-Taro Sekine, Tetsuro Yamashita, Hideyuki Takahashi. . . . . . . 97 [PDF]

Overwintering buds (OWBs) are dormant organs produced by perennial plants. After flowering, Japanese gentians (Gentiana triflora, G. scabra and their hybrids) produce OWBs in autumn and go dormant to survive winter. Although dormant OWBs have been reported to have strong cold and freezing tolerances, no details about mechanisms and regulation are known. We attempted to produce in vitro OWBs (IOWBs) from gentian plantlets to facilitate the study of OWBs. We found that high-sucrose (6%) culturing media induced IOWB formation. IOWBs showed an enhanced freezing tolerance together with high levels of proteins and transcripts know to specifically accumulate in OWBs. Furthermore, IOWBs showed endodormancy release and bud break in the same manner as OWBs. Bud breaking in OWBs and IOWBs was promoted by red light and inhibited by far-red light. These results indicated that IOWBs possess similar physiological characteristics as OWBs, at least regarding stress tolerance and dormancy. Therefore, our artificial IOWB induction technique will be useful for the study of physiological mechanisms in field-grown OWBs.

cDNA cloning and characterization of chalcone isomerase-fold proteins from snapdragon (Antirrhinum majus L.) flowers

Naoto Fujino, Tatsuya Yamazaki, Yanbing Li, Kota Kera, Erika Furuhashi, Satoshi Yamashita, Yasumasa Morita, Masayoshi Nakayama, Seiji Takahashi, Toru Nakayama. . . . . . . 105 [PDF]

cDNAs coding for the chalcone isomerase-fold proteins (CIFP) of the snapdragon (Antirrhinum majus L.) were cloned and characterized. One of these CIFPs was a Cluster-1 member of the CIFP family, which was a catalytically active chalcone isomerase and thus termed AmCHI1. The recombinant AmCHI1 could act on 2′,4,4′,6′-tetrahydroxychalcone (THC) and 2′,3,4,4′,6′-pentahydroxychalcone (PHC) to produce naringenin and eriodictyol with kcat/Km values of 0.25 s−1 μM−1 and 0.071 s−1 μM−1, respectively, at pH 7.5 and 4°C. The enzyme could not act on 4′-O-glucosides of THC and PHC. In the yellow snapdragon petals (cv. Yellow Butterfly), the temporal expression patterns of AmCHI1 were consistent with the observed temporal accumulation patterns of flavones. Thus, regulation of the AmCHI1 transcription and substrate specificity of the expressed AmCHI1 should serve as the key mechanisms that allows for partitioning of the flavonoid biosynthetic pathways into the aurones and the non-aurone flavonoids in snapdragon petal cells. The other CIFP cDNA, AmCIFP4, was a Cluster-4 member of the family and was similar in its primary structure to enhancers of the flavonoid production of torenia (Torenia×hybrida) and petunia (Petunia×hybrida). AmCIFP4 was more abundantly expressed than AmCHI1 irrespective of flower color.

Direct adventitious shoot organogenesis and plant regeneration from cotyledon explants in Neolamarckia cadamba

Hao Huang, JunCheng Li, KunXi OuYang, XianHai Zhao, Pei Li, BoYong Liao, XiaoYang Chen. . . . . . . 115 [PDF]

Neolamarckia cadamba is a fast-growing and deciduous tropical hardwood with anatomical, morphological, and chemical characteristics that make it suitable for building materials, pulp production, and medicine raw materials. In this study, a protocol for direct adventitious shoot organogenesis and plant regeneration from the aseptic cotyledons of N. cadamba was established. The cotyledons with petioles from 3-week-old seedlings were used for adventitious shoot induction in DCR medium containing 22.20 μM 6-benzyladenine (BA) and 0.27 μM α-naphthaleneacetic acid (NAA). The frequency of adventitious shoot induction was 54.2%. Micro-shoots were then transferred to MS medium containing 4.44 μM BA and 0.25 μM indole-3-butyric acid (IBA) for shoot propagation. Available shoots per explant reached 5.9. The highest rooting percentage (98.3%) was obtained when shoots were transferred to half-strength MS medium supplemented with 0.27 μM NAA and 0.25 μM IBA. The rooted plantlets could be successfully acclimatized to a greenhouse with more than 95% survival, and the regenerated plants showed the same morphological characteristics as those of the control plants in fields. Histological observations revealed that the adventitious shoots only originated from the epidermal tissue around the edge of the cut zone of the cotyledonary petiole.

VP16 fusion efficiently reveals the function of transcriptional repressors in Arabidopsis

Sumire Fujiwara, Keiko Kigoshi, Nobutaka Mitsuda, Kaoru Suzuki, Masaru Ohme-Takagi. . . . . . . 123 [PDF]

Proper gene expression regulated by transcription factors is essential for plants to achieve proper growth and development. However, the biological functions of many transcription factors remain largely unknown. Furthermore, although there are transcription factors which possess a plant-specific repression domain(s), their biological functions and whether such transcription factors function as transcriptional repressors are unclear. Thus, aiming for searching clues to understand their functions, we generated transgenic plants in which a putative transcriptional repressor fused with a VP16 viral trans-activation domain was expressed constitutively. Several plants with strong morphological phenotypes such as leaf and flower development defects were isolated from those lines expressing potential transcriptional repressors with unknown functions, giving the clue to reveal the yet-to-be analyzed functions of each protein. Reversal of function of the well-known transcriptional and floral repressor SHORT VEGETATIVE PHASE by VP16 fusion was observed, exemplifying successful functional reversion by this system. Plants constitutively expressing VP16 fused WUSCHEL, which is known to function both as a transcriptional activator and repressor, showed both phenotypes reported in its overexpression and loss-of-function lines. Taken together, our data provide examples showing the efficacy of VP16 fusion to provide helpful information to uncover the unknown functions of potential transcriptional repressors. This technique could also be effective to produce “super plants” which obtained strong and useful traits for application by strongly activating genes which are usually silent.

SDR6 is involved in regulation of owering time in Arabidopsis thaliana

Jihong Xing, Jing Zhang, Ping Yang, Chenxi Jiang, Jintao Fan, Jianmin Han, Jingao Dong. . . . . . . 133 [PDF]

Identification of flowering-time related genes in Arabidopsis thaliana has very important and meaningful to understand the regulation mechanism of the flowering-time. One late flowering mutant plant which displays a delayed transition to flowering was obtained from the Arabidopsis T-DNA insertion mutant library. SDR6, which encodes a short-chain dehydrogenase/reductase containing a NAD(P)-binding domain in Arabidopsis, has been identified as a novel flowering-time gene in Arabidopsis by further analyzing of SDR6 complementing plants. The sdr6 plants displayed later flowering than wild-type plants both in long and short days, but flowered later in short day than in long day. The flowering-time of SDR6 complementing plants is similar to that of wild-type plants. The late-flowering phenotype of sdr6 plants was reversed by gibberellin and vernalization treatments, which is similar to those of several mutants in the autonomous pathway. Compared with the wild-type, expression levels of FLC, LD, FVE, and SOC1 genes, key components of the autonomous pathway, were significantly altered in sdr6 mutants. However, expression levels of the key genes in the photoperiod, gibberellin, and vernalization pathways were not obviously different. Therefore, this gene may be involved in the autonomous flowering pathway to regulate the Arabidopsis flowering.

An improved tolerance to cadmium by overexpression of two genes for cysteine synthesis in tobacco

Michimi Nakamura, Tomoko Ochiai, Masaaki Noji, Yasumitsu Ogura, Kazuo T. Suzuki, Naoko Yoshimoto, Mami Yamazaki, Kazuki Saito. . . . . . . 141 [PDF]

Contamination of soil by heavy metals such as Cd causes a serious negative impact on agricultural production and human health. Thus, improvement of tolerance to Cd is one of the major challenges in plant biotechnology. In the present study, we have generated transgenic Nicotiana tabacum (tobacco) plants overexpressing both serine acetyltransferase (SAT) and cysteine synthase (CS) [O-acetylserine (thiol)-lyase], which are committed in the last two steps of cysteine (Cys) biosynthesis, by crossing the respective single-gene transgenic plants. Two enzymatic activities were high in these two-gene overexpressing plants, and these plants exhibited more resistance to Cd stress than wild-type and single-gene transgenic plants. The two-gene transgenic plants also exhibited a higher production of phytochelatins (PCs) in an inducible manner by the Cd stress. The levels of free non-chelated Cd were lower in the two-gene transgenic plants than the wild-type and single-gene transformants. The levels of Cys and γ-glutamylcysteine (γ-EC) were also increased in the dual transgenic plants, presumably enhancing the metabolic flow of Cys biosynthesis leading to the ultimate synthesis of PCs which detoxify Cd by chelating. These results suggested that the overexpression of two genes, SAT and CS, could be a promising strategy for engineering Cd resistant plants.

Regulatory change in cell division activity and genetic mapping of a tomato (Solanum lycopersicum L.) elongated-fruit mutant

Katarut Chusreeaeom, Tohru Ariizumi, Erika Asamizu, Yoshihiro Okabe, Kenta Shirasawa, Hiroshi Ezura. . . . . . . 149 [PDF]

Tomato elongated-fruit mutants have proven useful for elucidation and mapping of genes that regulate fruit elongation. We previously identified a novel tomato mutant, Solanum lycopersicum elongated fruit1 (Slelf1), which exhibits an elongated fruit shape caused by increased cell layers in the ovary proximal region, and located the causal genes on the long arm of chromosome 8. In this study, we isolated and characterized two independent tomato mutants, Solanum lycopersicum elongated fruit2 and 3 (Slelf2 and Slelf3). Histological analysis revealed that the Slelf2 mutant ovary displayed an overall elongated shape, whereas the developing ovary of the Slelf3 mutant was primarily expanded in the proximal region. We developed an intra-specific F2 population by crossing Slelf3 with Ailsa Craig, a related cultivar. Mapping of this population confirmed that the candidate gene of the Slelf3 mutation was positioned on the short arm of chromosome 7, in a region of approximately 0.4 Mb. These results indicate that the causal gene is a novel locus differing from previous genes known to affect fruit shape elongation in tomatoes. In addition, analysis of transcript levels of genes related to cell division and expansion in the Slelf3 mutant suggested that the mutated gene most likely regulates cell division activity, predominantly in the proximal region.

Arabidopsis glycerol-3-phosphate permease 4 is localized in the plastids and involved in the accumulation of seed oil

Hiromitsu Kawai, Toshiki Ishikawa, Toshiaki Mitsui, Shin Kore-eda, Maki Yamada-Kawai, Jun-ichi Ohnishi. . . . . . . 159 [PDF]

In plant cells, glycerol 3-phosphate (G3P) is apparently able to permeate the plastid envelope, but no specific transporter has been characterized so far. The Arabidopsis five G3Pp proteins have been predicted as putative G3P permeases because of their high homologies with the prokaryotic G3P/phosphate antiporter GlpT. In the present study, G3Pp4 was characterized in detail utilizing reverse genetic approaches. Promoter analysis using GUS expression revealed that G3Pp4 was expressed strongly throughout the embryos during late developmental stages, and the seed lipid contents decreased in two g3pp4 knockout mutants. An enhanced yellow fluorescent protein-fused G3Pp4 was localized in the plastids, functioned physiologically in A. thaliana, and had G3P-transport activity in E. coli. These results suggest that Arabidopsis G3Pp4 is a plastid envelope-localized G3P transporter and involved in accumulation of storage lipids in late embryogenesis.

Short Communication

Overexpression of SIZ1 enhances tolerance to cold and salt stresses and attenuates response to abscisic acid in Arabidopsis thaliana

Kenji Miura, Rieko Nozawa. . . . . . . 167 [PDF]

Sumoylation is a post-translational modification to control several cellular functions, including responses to environmental stresses and hormones in plants, and SIZ1 is a SUMO (small ubiquitin-related modifier) E3 ligase that plays an important role in sumoylation. In this study, we produced transgenic Arabidopsis thaliana plants with a moderately higher expression of SIZ1. Compared to wild type, the transgenic plants, with or without cold acclimation, were tolerant to freezing stress when subjected to freezing temperatures. The transgenic plants exhibited enhanced performance associated with robust shoot and root growth under salinity stress, whereas the wild type exhibited necrosis in the leaves with salt treatment. Furthermore, SIZ1 overexpression attenuated the root growth inhibition caused by abscisic acid. These results suggest that SIZ1 has the potential to genetically improve plant responses to abiotic stresses.

Arabidopsis xanthine dehydrogenase mutants defective in purine degradation show a compromised protective response to drought and oxidative stress

Shunsuke Watanabe, Yuka Kounosu, Hiroshi Shimada, Atsushi Sakamoto. . . . . . . 173 [PDF]

Although generally recognized for its role in nitrogen recycling and remobilization, purine catabolism might also be involved in the plant response and adaptation to environmental stress. Previously, we demonstrated that allantoin, a major purine intermediary metabolite in stressed plants, stimulates abscisic acid production and activates stress-responsive gene expression, leading to increased tolerance to abiotic stress in Arabidopsis seedlings. Here, we show that dysfunction of purine degradation, as a result of knocking down the key enzyme xanthine dehydrogenase, severely reduced the survival of Arabidopsis under progressive drought conditions and significantly decreased the tolerance to superoxide-mediated oxidative stress. The enhanced stress sensitivity of the knockdown mutants likely resulted from defective stress responses, because the drought-induced accumulation of the cellular protectant proline was compromised in the knockdown plants, which also showed lower mRNA levels of P5CS1, the gene encoding the rate-limiting enzyme for proline biosynthesis. When exogenously applied to wild-type Arabidopsis, allantoin and its precursor urate were able to elicit expression of P5CS1 in the absence of stress. Thus, our results provide further evidence for a previously unrecognized role for purine metabolites in stress responses, supporting the possible contribution of purine degradation to plant acclimation to changing environments.

An effcient plant regeneration through embryogenic callus formation and direct somatic embryogenesis via immature embryo culture in Ipomoea purpurea and I. tricolor

Hiroki Ishikuro, Eiji Nitasaka, Motoyasu Otani. . . . . . . 179 [PDF]

We established an efficient plant regeneration system to form embryogenic calli in common morning glory (Ipomoea purpurea) and blue morning glory (I. tricolor). Immature embryos of both morning glories cultured on media containing 1 mg l−1 4-fluorophenoxyacetic acid (4FA) and 6% sucrose formed many embryogenic calli. The frequency of embryogenic callus formation was highest in I. purpurea strain Q74 (42.5%) and in I. tricolor cultivar ‘Flying Saucers’ (36.7%). Embryogenic callus formation differed with the genotypes in both morning glories. Numerous somatic embryos were formed from the embryogenic calli when the calli were transferred onto plant growth regulator-free medium.

Note

EMS mutagenesis and characterization of Brassica rapa mutants

Yosuke Mizuno, Shunsuke Okamoto, Miyuki Hara, Tsuyoshi Mizoguchi. . . . . . . 185 [PDF]

Brassica rapa is an important model crop in the genus Brassica, which includes various important vegetable crops such as Chinese cabbage and turnip, and is closely related to Arabidopsis thaliana (Arabidopsis). B. rapa var. trilocularis (yellow sarson), a rapid-cycling population of B. rapa, is commonly used for genetic research and is expected to bridge the gap between the model plant Arabidopsis and Brassica crops. In this study, 940 M2 mutagenized lines of yellow sarson were produced using ethylmethanesulfonate (EMS) mutagenesis, and these M2 plants were examined for alterations in visible phenotypes. In total, 293 independent M2 lines (including 2188 M2 plants) were investigated for phenotypic alterations, and 394 individual mutants were isolated. Subsequently, the observed mutant phenotypes were classified into 8 major categories and 18 subcategories. In addition, three mutants, namely, early flowering 1 (elf1), crane-like 1 (crl1), and rosette 2 (ros2), were selected for further phenotypic and genetic analysis. elf1 and ros2 showed early and late flowering phenotypes, respectively. Based on their phenotypes, crl1 and ros2 are likely mutations in genes for auxin signaling and gibberellic acid biosynthesis, respectively.

The longer version of Arabidopsis thaliana heat shock protein 18.2 gene terminator contributes to higher expression of stably integrated transgenes in cultured tobacco cells

Takeshi Matsui, Kazutoshi Sawada, Eiji Takita, Ko Kato. . . . . . . 191 [PDF]

In order to achieve higher expression of a transgene, it is important to optimize not only the promoter, 5′-untranslated region (UTR), and localization signal, but also the transcriptional terminator. We previously reported that the terminator derived from the Arabidopsis thaliana heat shock protein 18.2 gene (HSPT) increases gene expression in various plant species. In this study, with the goal of further increasing the expression level of transgenes, we evaluated a longer version of HSPT, corresponding to the 878 bp downstream of the stop codon, in cultured tobacco cells. The longer version of HSPT increased the expression levels of various genes including Renilla reniformis luciferase, Photinus pyralis luciferase, horseradish (Armoracia rusticana) peroxidase, and the non-toxic B subunit of Stx2e, a candidate vaccine protein for pig edema disease. This effect was not observed in a transient expression system. This element represents a useful tool for expression of transgenes integrated into the nuclear genome in plant cells.