Plant Biotechnology Vol.33 No.2


Original Papers

Cortical microtubule disorganized related to an endogenous gibberellin increase plays an important role in rice mesocotyl elongation

Qian Liang, Che Wang, Dianrong Ma, Li Li, Zhibo Cui, Xiaoxue Wang, Qian Qian, Baodong Cai, Yuqi Feng, Wenfu Chen. . . . . . . 59 [PDF]

Mesocotyl elongation in rice is essential for seedling emergence. Our previous screening identified two weedy rice accessions (Oryza sativa f. spontanea L.) (WR04-6 and WR04-43) were with unusually long mesocotyl. In this study, using rice cultivar (Nipponbare, Oryza sativa L., subspecies temperate japonica) as a control, we found that at the middle and end of mesocotyl elongation, the predominant cortical microtubules (CMTs) in weedy rice are transversely oriented that related to cell elongation, while, cultivated rice had oblique MT arrays. Interestingly, more extensive MT depolymerization was observed in the early stage of mesocotyl elongation in weedy rice accessions than that in cultivated rice. Moreover, MT-destabilizing agent oryzalin treatment (25 nM) led to an increase of rice mesocotyl length, which is correlated with mesocotyl cell elongation. On the contrary, MT-stabilizing agent taxol led to a full inhibition of weedy rice mesocotyl growth. Further, oryzalin treatments resulted in an endogenous gibberellin (GA) increase and the high expression of GA biosynthesis related gene-GA20ox2, GA3ox2, and GIBBERELLIN-INSENSITIVE DWARF1 (GID1), and taxol treatments had an opposite effect on the change of GA content and the GA biosynthesis gene expression. By contrast, the early CMT depolymerization in the weedy rice mesocotyl elongation was not affected by the change of GA content. Our findings suggested that an early depleted form of CMTs is a main reason of unusually long mesocotyls in weedy rice, and it plays an important role in rice mesocotyl growth, which has a close relationship with an increase of endogenous GAs.

AINTEGUMENTA negatively regulates age-dependent leaf senescence downstream of AUXIN RESPONSE FACTOR 2 in Arabidopsis thaliana

Guanping Feng, Qiang Xu, Zhenyang Wang, Qiangjiu Zhuoma. . . . . . . 71 [PDF]

Leaf senescence is the final stage of leaf development and is regulated by many internal and external cues. As a repressor of auxin signaling, AUXIN RESPONSE FACTOR 2 (ARF2) is involved in control of several developmental processes, but its functional mechanism on how to positively regulate leaf senescence has not been clearly defined. Here, we examined AINTEGUMENTA (ANT), a member of the AP2/ERF transcription factor family, and found that ANT played an important role in extending leaf longevity. The loss-of-function ant-1 mutant showed premature leaf senescence, whereas overexpression of ANT led to a delay in leaf senescence. Genetic analysis revealed that loss of ANT repressed the delayed leaf senescence phenotype in arf2-5 mutant. Taken together, our results suggest that ANT is involved in regulation of leaf senescence downstream of ARF2.

Arabidopsis AtRRP44 has ribonuclease activity that is required to complement the growth defect of yeast rrp44 Mutant

Naoyoshi Kumakura, Hiroka Otsuki, Masaru Ito, Mika Nomoto, Yasuomi Tada, Kunihiro Ohta, Yuichiro Watanabe. . . . . . . 77 [PDF]

The RNA exosome is a multiprotein complex responsible for 3′ to 5′ degradation and processing of various classes of RNAs in eukaryotes. Rrp44/Dis3 is the catalytic center of the RNA exosome in yeast and human. Previously, we identified Arabidopsis thaliana RRP44 (AtRRP44) as a single functional homolog of Rrp44/Dis3. Although AtRRP44 is potentially a catalytic center of the plant RNA exosome, the ribonuclease activity of AtRRP44 has not been tested. Here, we show that AtRRP44 has ribonuclease activity using in vitro translated recombinant proteins. Mutation of the aspartic acid residue D489 of AtRRP44 to asparagine (D489N) resulted in loss of ribonuclease activity, indicating that aspartic acid is at the active site. The wild-type AtRRP44 protein rescued the growth defect of Saccharomyces cerevisiae rrp44 mutants, but the D489N mutated AtRRP44 did not. This finding suggests that the ribonuclease activity of wild-type AtRRP44 is required for yeast cell viability. We also showed that AtRRP44 was highly expressed in organs experiencing active cell turnover, such as shoot apical meristem, root apical meristem, and lateral root primordium. Along with previous studies showing that loss of RRP44 in Arabidopsis is lethal, our results suggest that AtRRP44 has ribonuclease activity that is related to plant development.

Strigolactones are involved in sugar signaling to modulate early seedling development in Arabidopsis

Guo Dong Li, Li Na Pan, Kai Jiang, Ikuo Takahashi, Hidemitsu Nakamura, Ying Wu Xu, Tadao Asami, Ren Fang Shen. . . . . . . 87 [PDF]

Strigolactones are a novel class of plant hormones that interact with multiple signaling molecules, including auxin, abscisic acid, ethylene, and brassinosteroid, to regulate plant growth and development. Recently, researchers have shown that sugars are involved in bud outgrowth control, suggesting a potential interaction between sugars and strigolactone signaling. To better understand the relationship between strigolactones and sugar in plant development, the sugar sensitivity of strigolactone biosynthesis and signaling mutants (max1 and max2) was evaluated in early seedling development with a low-glucose assay. Both max1 and max2 displayed obvious hyposensitivity to glucose repression, as do gin mutants, but they were hypersensitive like the wild type to the high-glucose conditions used for gin mutant screening. The strigolactones acted synergistically with glucose in repressing seedling establishment. A further comparative transcriptomic analysis indicated that the expression of stress-related genes in the max2 mutant is impaired by glucose, and a carbohydrate analysis revealed a reduced hexose content in the max mutants. Our results suggest that the roles of strigolactones in the regulation of early seedling development are probably independent of the HXK1 signaling pathway. Taken together, these findings provide evidence that strigolactones are involved in sugar signaling, thus modulating early seedling development.

Sweet potato expressing the rice Zn transporter OsZIP4 exhibits high Zn content in the tuber

Youngsup Shin, Ryuichi Takahashi, Hiromi Nakanishi, Takashi Yamakawa. . . . . . . 99 [PDF]

Zinc (Zn) is an essential element for humans as well as for plants. Sweet potato is a major staple crop in the world, and is an attractive target crop for genetic engineering because its suitability for clonal propagation and its self-incompatibility. OsZIP4 is a rice Zn transporter that increases Zn concentrations, especially in roots, when it is artificially overexpressed in rice. In our study we induced an embryogenic callus from the apex of sweet potato (Ipomoea batatas L. Lam. cv. Kokei No. 14), and obtained OsZIP4 overexpressed lines driven by the CaMV35S promoter through Agrobacterium infection. Three transgenic lines were confirmed by RT-PCR for their OsZIP4 expression in roots and leaves. Zn concentrations in roots and leaves of OsZIP4 transgenic plants grown on LS medium were approximately 2.3 times and 1.3 times higher, respectively than those of wild type plants. In addition, the iron content of tubers was two times higher than wild type (WT) lines, and that of leaves was up to 1.2 times higher. Root tubers of transgenic lines were obtained under soil culture. The Zn concentration of transgenic sweet potato tubers was 2.2 times higher than WT lines, but there were no differences in shoot length or fresh weight between the transgenic and WT lines. These results indicate that introducing the OsZIP4 gene in to sweet potato is a potential method to improve the Zn nutrition of livestock and humans.

Difference of saccharification yields between organs and growth stages in rice

Tomomi Abe, Ryota Iiyoshi, Kyosuke Ito, Yutaka Takahashi, Kohei Sato, Atsushi Matsuzaka, Tomonori Sonoki, Yukihiro Ito. . . . . . . 105 [PDF]

Saccharification is a key step in the efficient production of biofuels and biomaterials from cellulosic biomass. We examined saccharification yields from leaf blades, leaf sheaths and stems at several growth stages in rice. We found that saccharification yields were high before heading and reduced after heading in all three organs examined. Stems showed highest saccharification yields at all growth stages examined, and leaf blades showed lowest saccharification yields. Differences of saccharification yields between rice cultivars were also observed. Our results indicate that saccharification yields are different between rice organs. This suggests that the proportion of organs is one of the determinants of saccharification yields of rice straws, and thus it will be a breeding target for biofuel and biomaterial crops with high saccharification yields. Our results also suggest that the harvesting stage is critical for high saccharification yields.

Short Communication

Silencing of Nicotiana benthamiana SEC14 phospholipid transfer protein reduced jasmonic acid dependent defense against Pseudomonas syringae

Akinori Kiba, Yu Imanaka, Masahito Nakano, Ivan Galis, Yuko Hojo, Tomonori Shinya, Kouhei Ohnishi, Yasufumi Hikichi. . . . . . . 111 [PDF]

We previously identified SEC14, phospholipid transfer protein superfamily gene, in Nicotiana benthamiana (NbSEC14) that was closely related to phospholipid signaling as well as jasmonic acid-dependent defense responses during plant immune responses against Ralstonia solanacearum. To examine effect of NbSEC14-silencing on basal plant defenses, we used two other bacterial pathogens with different virulent strategies, Pseudomonas syringae pv. tabaci and pv. mellea. NbSEC14-silenced plants showed accelerated growth of P. syringae pv. tabaci and pv. mellea, and formation of necrotic lesions. Induction of JA-related PR-4 gene was compromised in NbSEC14-silenced plants, which was supported by reduced jasmonic acid levels in NbSEC14-silenced plants. These results suggested that NbSEC14 might be regulating plant basal resistance against plant pathogenic Pseudomonads via jasmonic acid-dependent signaling pathway.


A fluorometric assay for quantification of fucoidan, a sulfated polysaccharide from brown algae

Yoshie Yamazaki, Yuko Nakamura, Tatsuo Nakamura. . . . . . . 117 [PDF]

Brown algae contain a fucose-rich sulfated polysaccharide called fucoidan which is a constituent of the extracellular matrix. In recent years, fucoidan has attracted much attention for its pharmaceutically important biological activities. To develop a superior method for fucoidan quantification, we investigated several cationic dyes with potential interaction with the negatively charged sulfate groups of fucoidan and found that a commercially available fluorescent dye, SYBR Gold nucleic acid gel stain, showed a clear fluorescence enhancement upon mixing with authentic fucoidan. A standard curve generated with the dye and authentic fucoidan showed a linear dynamic range of 0.05–2.5 ng µl−1, and the limits of detection and quantification of fucoidan were 0.025 and 0.075 ng µl−1, respectively. Neither nucleic acids nor alginates, anionic polymers present in brown algae, interfered with the quantification of fucoidan, which is simply extracted with diluted HCl from sporophytes of the brown alga Saccharina japonica. The fluorometric microplate method in this study is simple, sensitive, and widely applicable to fucoidan quantification.

Identification of a xylem sap germin-like protein and its expression under short-day and non-freezing low-temperature conditions in poplar root

Tsutomu Aohara, Hiroaki Mizuno, Daiki Kiyomichi, Yuta Abe, Kaoru Matsuki, Keiko Sagawa, Hitoshi Mori, Hiroaki Iwai, Jun Furukawa, Shinobu Satoh. . . . . . . 123 [PDF]

In the shoots of photoperiod-sensitive deciduous trees, including poplar, short-day and non-freezing low-temperature conditions induce bud dormancy and its break, respectively, and these conditions also induce shoot cold acclimation. In a previous study, levels of organic and inorganic components, including proteins, increased in the xylem sap of Populus nigra in winter, suggesting seasonal changes in root functions. Here, analysis of a major xylem sap protein (XSP24) of P. nigra in winter by mass spectrometry together with the whole genome sequence of P. trichocarpa and transcript abundance in roots under short-day conditions identified PtXSP24 to be a germin-like protein of the cupin superfamily, which was reported to be associated with various stresses and to have oxalate oxidase and/or superoxide dismutase activities in the cell wall. Expression of XSP24, which corresponds to PtXSP24 in P. maximowiczii, a potentially useful Japanese native poplar in the same phylogenetic clade as P. trichocarpa, was enhanced under short-day and non-freezing low-temperature conditions, as well as by application of abscisic acid. These results suggest that XSP24 is involved in tolerance to environmental stresses in autumn and early winter.

The Arabidopsis CAPRICE protein fused to the VP16 transcriptional activation domain alters root hair and trichome development

Rumi Tominaga-Wada, Takuji Wada. . . . . . . 129 [PDF]

CAPRICE (CPC) is a key regulator of epidermal cell fate determination, including root hair and trichome formation, in Arabidopsis thaliana. CPC encodes an R3 MYB transcription factor and is known to be involved in the transcriptional repression of the downstream gene GLABRA2 (GL2). We examined transgenic plants harboring CPC fused to a virus-derived transcriptional activator domain (CPC:VP16). Plants carrying 35S::CPC:VP16 showed increased root hair formation, similar to 35S::CPC plants, compared to the wild-type plants. However, transgenic plants harboring CPC:VP16 under the control of CPC promoter (CPC::CPC:VP16) showed similar root hair phenotype to that of the wild-type plants, suggesting an inherent cell-to-cell movement ability of CPC:VP16. In this study, all transgenic plants harboring CPC:VP16 possessed a reduced number of trichomes, nearly identical to that of 35S::CPC plants, compared to the wild type. Furthermore, we observed some unusual tissues with ectopic trichome clusters in all transgenic plants harboring CPC:VP16. These results indicate that VP16 generally does not confer the transcriptional activation ability to CPC.

Modification of the filter-inserted tip method for simple and rapid DNA extraction from strawberry

Manabu Watanabe, Masanobu Fukami. . . . . . . 133 [PDF]

In order to perform conveniently DNA extraction from strawberry leaves, we tried to apply the “filter-inserted tip method”. This method has been used to extract easily DNA from rice. To examine the utility of the method in strawberry leaves, DNA extraction experiment was carried out, and the yielded DNA samples were used for PCR reactions. PCR reaction was greatly improved with the crushing buffer when 50 mg/ml polyvinylpolypyrrolidone was added. PCR amplicons were obtained for 132–140 out of 144 samples examined, showing high reproducibility of the method. It took approximately 70 min to conduct DNA extractions from 24 samples. It was about 40% of the operation time in the case of using a commercially available DNA extraction kit. The results suggest that this method is suitable for rapid DNA extractions from a large number of samples for PCR reactions, such as marker-assisted selection with simple manipulations, low cost, and without using expensive equipment.

Transformation and isoflavonoid analyses of suspension-cultured cells of soybean [Glycine max (L.) Merr. cv. Enrei]

Toshiyuki Waki, Seiji Takahashi, Toru Nakayama. . . . . . . 137 [PDF]

We established cell suspension cultures of soybean [Glycine max (L.) Merr.] (cv. Enrei). Calli were induced from cotyledon explants on callus induction medium containing 1 mgl−1 kinetin and 0.1 mgl−1 2,4-dichlorophenoxyacetic acid (2,4-D) and then successfully suspension-cultured in Murashige and Skoog basal medium containing 1 mgl−1 kinetin and 0.05 mg l−1 2,4-D. The resulting suspension-cultured cell line, designated as ECW1, has been maintained for 3 years by subculturing at 8-day intervals. Flavonoids that accumulated in ECW1 cells during cultivation were analyzed by high performance liquid chromatography. These analyses showed that 6″-O-malonyldaidzin was the most abundant flavonoid until day 8 of cultivation, followed by 6″-O-malonylgenistin, daidzin, and genistin. Trace amounts of isoflavone aglycons, glyceollin, and other flavonoids were detected during cell cultivation. We established an Agrobacterium-mediated method to transform suspension-cultured soybean cells using the ECW1 cell line as the host. A gene encoding green fluorescent protein was heterologously expressed under the control of the CaMV 35S promoter in ECW1 cells. The recombinant cells were stably maintained for 2.5 years via repeated subculturing. This is the first example of the stable transformation of suspension-cultured soybean cells using the Agrobacterium method. The cell line ECW1 is a promising tool for research on the biochemistry, molecular biology, and physiology of soybean.