Plant Biotechnology Vol.35 No.3


Original Papers

The possible roles of AtERF71 in the defense response against the Fusarium graminearum

Fitri Yelli, Tomoaki Kato, Takumi Nishiuchi . . . . . . . 187 [PDF] [Supplement]

The ethylene (ET) signaling pathway is involved in plant immunity and contributes to the disease tolerance of plants to necrotrophic phytopathogens. Ethylene response factors (ERFs) are known to play important roles in the transcriptional regulation of defense genes by ET. In the present study, we analyzed the function of AtERF71 belonged to group VII ERF family in disease resistance against a hemibiotrophic fungal phytopathogen, Fusarium graminearum. When conidia solutions were dropped onto intact leaves of Arabidopsis plants, both ein2-1 and ein3-1 mutants showed enhanced disease resistance against F. graminearum compared with the wild type. This finding suggested that the ET signaling pathway was involved in the resistance to Fusarium entry into the leaf epidermis in Arabidopsis plants. We discovered that the AtERF71 expression was significantly induced by inoculation with F. graminearum. This induction of AtERF71 was suppressed in the ein3-1 mutant. Enhanced disease resistance was observed in the leaves of the aterf71 mutant when compared with wild type. In addition, the expression levels of the JA/ET-responsive PDF1.2 gene were significantly downregulated in the aterf71 mutant after inoculation with F. graminearum. Taken together, these results indicate the possible involvement of AtERF71 in disease tolerance to F. graminearum in Arabidopsis plants.

The validity of carbon isotope discrimination as a screening criterion for grain yield in two barley landraces under deficit irrigation with saline water in southern Tunisia

Mohamed Bagues, Behrooz Sarabi, Jaleh Ghashghaie, Ikbel Souli, Kamel Nagaz . . . . . . . 193 [PDF] [Supplement]

Arid and semiarid regions with rain shortage and scarce good quality water must make use of low-quality water for irrigation. Consequently, improved plant cultivars for use in these areas should show adaptation capacities to confer drought and salt resistance and allow the cultivation under limited water availabiltiy. The present study was conducted to determine the effect of deficit irrigation with saline water on two local barley landraces, “Karkeni” and “Bengardeni”. Plants were saline-irrigated with three watering regimes during tillering, heading, and grain filling stages. Biochemical traits, carbon isotope discrimination (Δ13C), mineral composition, grain yield (GY) and water use efficiency based on grain yield (WUEgy) were evaluated as performance indicators. Almost all of the studied traits (e.g. soluble carbohydrates, proline, Δ13C, Na concentration, and GY) were significantly affected by deficient saline-irrigation regimes at different growth stages. The hierarchical clustering analysis clearly showed that Δ13C placed very close to GY averaging two barley landraces, which was in accordance with the scatter plot result. Multiple linear regression performed between GY as the dependent variable and other traits studied as the independent variables indicated that WUEgy, Δ13C, and soluble carbohydrates significantly explained the variability in GY (R2=95.64%). A significant positive correlation that observed between Δ13C and GY at three growth stages, indicated that Δ13C may be an important proxy component for indirect selection of yield potential in barley under deficient irrigation regimes with saline water. According to our result, “Karkeni” seems to be more efficient in terms of higher GY, WUEgy, proline and carbohydrate contents, K, Mg and Zn concentrations, as well as lower Δ13C and lipid peroxidation as compared with “Bengardeni”, under low osmotic potential imposed by deficient irrigation treatments with saline water, “Karkeni” can thus be selected and used as a parent in order to obtain more tolerant plants against such stresses in future breeding programs.

Expression and functional analysis of apple MdMADS13 on flower and fruit formation

Masato Wada, Hidemi Oshino, Norimitsu Tanaka, Naozumi Mimida, Yuki Moriya-Tanaka, Chikako Honda, Toshio Hanada, Hiroshi Iwanami, Sadao Komori . . . . . . . 207 [PDF]

Apple MdMADS13 has a transcription factor with MADS domain. Moreover, it is expressed specifically at petals and carpels. The product forms a dimer with MdPISTILLATA (MdPI) protein as a class B gene for floral organ formation. Reportedly, in parthenocarpic cultivars of apple (Spencer Seedless, Wellington Bloomless, Wickson and Noblow) the MdPI function is lost by genome insertion of retrotransposon, which cultivars show a homeotic mutation of floral organs, petals to sepals and stamens to carpels. Apple fruit is pome from receptacle tissue, and MdSEPALLATA (MdMADS8/9) and AGAMOUS homologues MdMADS15/22 involved in the fruit development, the transgenic apple suppressed these gene showed poor fruit development and abnormal flower formation. This article describes that the MdMADS13 retained expression after blossom and small fruits of parthenocarpic cultivars. Yeast two-hybrid experiment showed specific binding between MdPI and MdMADS13 proteins. Furthermore, transgenic Arabidopsis with 35S::MdMADS13 have malformed stamens and carpels. These results suggest strongly that MdMADS13 is related to flower organ formation as a class B gene with MdPI.

Transcriptional enhancement of a bacterial choline oxidase A gene by an HSP terminator improves the glycine betaine production and salinity stress tolerance of Eucalyptus camaldulensis trees

Ngoc-Ha Thi Tran, Taichi Oguchi, Etsuko Matsunaga, Akiyoshi Kawaoka, Kazuo N. Watanabe, Akira Kikuchi . . . . . . . 215 [PDF] [Supplement]

Novel transgenic Eucalyptus camaldulensis trees expressing the bacterial choline oxidase A (codA) gene by the Cauliflower mosaic virus (CaMV) 35S promoter and the Arabidopsis thaliana heat shock protein (HSP) terminator was developed. To evaluate the codA transcription level and the metabolic products and abiotic stress tolerance of the transgenic trees, a six-month semi-confined screen house cultivation trial was conducted under a moderate-stringency salt-stress condition. The transcription level of the CaMV 35S promoter driven-codA was more than fourfold higher, and the content of glycine betaine, the metabolic product of codA, was twofold higher, with the HSP terminator than with the nopaline synthase (NOS) terminator. Moreover, the screen house cultivation revealed that the growth of transgenic trees under the salt stress condition was alleviated in correlation with the glycine betaine concentration. These results suggest that the enhancement of codA transcription by the HSP terminator increased the abiotic stress tolerance of Eucalyptus plantation trees.

Evaluation of internal control genes for quantitative realtime PCR analyses for studying fruit development of dwarf tomato cultivar ‘Micro-Tom’

Seung-won Choi, Ken Hoshikawa, Satoshi Fujita, Dung Pham Thi, Tsuyoshi Mizoguchi, Hiroshi Ezura, Emi Ito . . . . . . . 225 [PDF] [Supplement]

Quantitative real-time PCR (qRT-PCR) is widely used to analyze the expression profiles of the genes of interest. In order to obtain accurate quantification data, normalization by using reliable internal control genes is essential. In this study, we evaluated the stability and applicability of eight internal control gene candidates for analyzing gene expression during fruit development in dwarf tomato cultivar Micro-Tom. We collected seventeen different samples from flowers and fruits at different developmental stages, and estimated the expression stability of the candidate genes by two statistical algorithms, geNorm and NormFinder. The combined ranking order and qRT-PCR analyses for expression profiles of SlYABBY2a, SlYABBY1a, FRUITFULL1 and APETALA2c suggested that EXPRESSED was the most stable and reliable internal control gene among the candidates. Our analysis also suggested that RPL8 was also suitable if the sample group is limited to fruits at different maturation stages. In addition to EXPRESSED, GAPDH was also applicable for relative quantitation to monitor gene expression profiles through fruit development from pistil to pericarp.

The effects of Lepidopteran oral secretion on plant wounds: A case study on the interaction between Spodoptera litura and Arabidopsis thaliana

Natsuko Kinoshita, Shigeyuki Betsuyaku . . . . . . . 237 [PDF]

This paper is about the cellular responses of plants to chewing insect attacks. We deployed a recently developed experimental system to monitor the responsiveness of Arabidopsis thaliana (Arabidopsis) to the application of oral secretion (OS) from Lepidopteran generalist herbivore Spodoptera litura (S. litura). Oral secretion from S. litura contains gut regurgitant and saliva. We identified significant differences in the wound closure morphologies (e.g., dried and sealed tissue) between mechanically damaged leaves with and without an application of S. litura OS at the site-of-injury. Experimental controls were mechanically wounded leaves. Wounds were walled off by visible vertical cross sections. Cell death was restricted to the immediate areas of the wounds. In contrast, mechanically damaged leaves treated with S. litura OS did not display a clear sealing pattern due to an absence of a defined vertical cross section at the wound site. Notably, OS treated leaves exhibited a wider area of visible premature senescence (the declining of chlorophyll content caused by death of chloroplasts) around the injury than controls. More pronounced senescence was also observed around the injury in S. litura OS treated wounds than in controls. Heat inactivated S. litura OS elicited a similar response to non-heat inactivated samples. The causal compound is heat stable and thus not a protein. Our results suggest that S. litura OS: (1) inhibited wound recovery responses in leaves; (2) promoted senescence around injured areas. The function of senescence may be to relocate nutritional resources to support plant survival when attacked.

Dissecting promoter of InMYB1 gene showing petal-specific expression

Mirai Azuma, Yoshimi Oshima, Shingo Sakamoto, Nobutaka Mitsuda, Masaru Ohme-Takagi, Shungo Otagaki, Shogo Matsumoto, Katsuhiro Shiratake . . . . . . . 243 [PDF]

We had previously reported that the InMYB1 promoter, the 1023 bp upstream region of InMYB1, works petalspecifically in various dicot plants by recognizing petal identity at a cellular level. To determine the petal-specific region in the InMYB1 promoter, Arabidopsis plants harboring InMYB1_1023b::GUS (β-glucuronidase), InMYB1_713b::GUS, InMYB1_506b::GUS, InMYB1_403b::GUS, InMYB1_332b::GUS, InMYB1_200b::GUS and InMYB1_140b::GUS were produced and confirmed a shortest region, which has the petal-specific promoter activity by using histochemical GUS assay. Petal-specific GUS staining was not observed in the Arabidopsis plants transformed with InMYB1_200b::GUS and InMYB1_140b::GUS, but observed in transgenic Arabidopsis plants harboring from InMYB1_1023b::GUS to InMYB1_332b::GUS. cDNA sequence of InMYB1 shows that 120 bp upstream region of InMYB1 is 5′ untranslated region suggesting that the 332-121 bp upstream region of InMYB1 contains an important element for petal-specific gene expression. In the Arabidopsis harboring the InMYB1_332-121b×3_TATA_Ω::GUS, petal-specific GUS staining was observed and the staining was stronger than in the Arabidopsis harboring InMYB1_1023b::GUS. This result shows that the 332-121 bp region is enough and essential for the petal specificity and the InMYB1_332-121b×3_TATA_Ω could be used for the molecular breeding of floricultural crops.

Biological effects of ion beam irradiation on perennial gentian and apple

Nobuhiro Sasaki, Aiko Watanabe, Tomonori Asakawa, Makoto Sasaki, Nobue Hoshi, Zenbi Naito, Yoshiya Furusawa, Takashi Shimokawa, Masahiro Nishihara . . . . . . . 249 [PDF]

The development of new varieties of perennial plants generally requires lengthy and laborious procedures. In this study, we used ion beam irradiation mutagenesis in an attempt to accelerate the breeding process for perennial plants. We evaluated the biological effects of five ion beam sources (carbon, neon, argon, silicon, and iron) and neutron irradiation on Japanese gentian and apple. These treatments were applied at the National Institute of Radiological Sciences (NIRS) using the Heavy Ion Medical Accelerator in Chiba (HIMAC) and the Neutron-exposure Accelerator System for Biological Effect Experiments (NASBEE). Biological effects were observed in in vitro gentian plants after irradiation with ion beams at <10 Gy, whereas apple trees were less sensitive to ion beam irradiation. The growth of gentians in vitro was repressed by 3 Gy neutron irradiation, while that of grafted apple trees was not affected by 4 Gy neutron irradiation. During in vitro proliferation, seven pink-flowered lines were obtained from originally blue-flowered gentian after C and Ne ion beam irradiation treatments. Genomic and reverse transcription-PCR analyses of these lines suggested that the mutations occurred in the genomic region containing F35H (encoding flavonoid 3′,5′-hydroxylase). These results provide useful information for the mutagenesis and breeding of gentian, apple, and other perennial plants.

Anthocyanin mutants of Japanese and common morning glories exhibit normal proanthocyanidin accumulation in seed coats

Kyeung Il Park, Eiji Nitasaka, Atsushi Hoshino . . . . . . . 259 [PDF]

Anthocyanin and proanthocyanidin biosynthesis pathways are believed to overlap. This study examined proanthocyanidin accumulation in seed coats of morning glories (Ipomoea nil and I. purpurea) carrying mutations in CHS-D, CHI, and ANS genes encoding chalcone synthase, chalcone isomerase, and anthocyanidin synthase, respectively. Chemical staining revealed that mutants accumulate proanthocyanidin normally. Thus, the tested genes are not essential to proanthocyanidin biosynthesis, but are essential to anthocyanin biosynthesis in flowers and stems. Based on the results and the I. nil draft genome sequence, the genes involved in proanthocyanidin biosynthesis, including a new copy of the flavanone 3-hydroxylase gene could be predicted. Moreover, the genome has no homologs for known enzymes involved in producing flavan-3-ols, the starter and extension units of proanthocyanidin. These results suggested that I. nil produces flavan-3-ols through an undiscovered biosynthesis pathway. To characterize proanthocyanidin pigmentation further, we conducted mutant screening using a large I. nil population. We discovered that the brown mutant lines (exhibiting brown seeds and normal anthocyanin pigmentation) do not accumulate proanthocyanidin in their seed coats. Thus, the brown mutation should be useful for further investigations into the various mechanisms controlling anthocyanin and proanthocyanidin pathways.

Cytokinin-induced expression of OSH1 in a shoot-regenerating rice callus

Masashi Naruse, Honami Takahashi, Nori Kurata, Yukihiro Ito . . . . . . . 267 [PDF]

The expression of a KNOX class 1 gene OSH1 is induced by cytokinin during regeneration of shoots from callus in Oryza sativa L. (rice). This cytokinin-induced expression was enhanced by overexpression of homologues of cytokininsignalling phosphorelay genes such as a histidine kinase gene OHK3, a phosphotransmitter gene OHP2 and a response regulator gene ORR1 in cultured cells. Regionally overlapped expression of these genes and OSH1 was observed in shoot apex. These results suggest that these cytokinin-signalling genes are positive regulators of the expression of OSH1, and mediate the OSH expression upon shoot regeneration from callus in rice.

Short Communication

Overexpression of OsAP2 and OsWRKY24 in Arabidopsis results in reduction of plant size

Seonghoe Jang, Hsing-Yi Li . . . . . . . 273 [PDF] [Supplement1] [Supplement2]

Recently, two rice genes, OsAPETALA2 (OsAP2) and OsWRKY24 have been reported to be positive regulators involved in increased lamina inclination and grain size through cell elongation. Here, we found that the two genes have tightly linked expression patterns and functional convergence in rice, and are also likely to play an opposite role in Arabidopsis. Overexpression of the two rice transcription factors in Arabidopsis caused smaller plant size with reduced cell size, and the expression of a series of genes encoding expansins and xyloglucan endotransglucosylase/hydrolases (XTHs) involved in cell elongation was reduced. However, transgenic Arabidopsis expressing OsWRKY24-SRDX as a synthetic chimeric repressor displayed indistinguishable phenotypes from wild-type plants. Moreover, the subcellular localization pattern of OsWRKY24 in Arabidopsis was different from that in rice. Thus, we demonstrate an example of transcription factors from one species playing distinct roles in different plant species.


Salinity stress-responsive transcription factors in the liverwort Marchantia polymorpha

Hiroyuki Tanaka, Rin Suzuki, Nanako Okabe, Tomohiro Suzuki, Yutaka Kodama . . . . . . .  . . . 281 [PDF] [Supplement1] [Supplement2] [Supplement3] [Supplement4] [Supplement5] [Supplement6] [Supplement7] [Supplement8]

Salinity stress limits plant growth and productivity. To cope with this limitation, the expression patterns of numerous genes are altered in response to salt stress; however, the regulatory mechanisms involved in these changes are unclear. In the present study, we investigated the regulation of the salinity stress response in the liverwort Marchantia polymorpha. The growth of M. polymorpha gemmalings was severely inhibited by NaCl, and RNA-sequencing and quantitative RT-PCR analyses revealed that the expression of several transcription factor gene families was induced by salinity stress. This work provides insight into the molecular mechanisms underlying the salinity stress response in M. polymorpha.

An NAC domain transcription factor ATAF2 acts as transcriptional activator or repressor dependent on promoter context

Isura Sumeda Priyadarshana Nagahage , Shingo Sakamoto, Minoru Nagano, Toshiki Ishikawa, Maki Kawai-Yamada, Nobutaka Mitsuda, Masatoshi Yamaguchi . . . . . . . 285 [PDF] [Supplement]

The ARABIDOPSIS THALIANA ACTIVATION FACTOR 2 (ATAF2) protein has been demonstrated to be involved in various biological processes including biotic stress responses, photo morphogenesis, and auxin catabolism. However, the transcriptional function of ATAF2 currently remains elusive. Therefore, to further understand the molecular function of ATAF2, we evaluated the transcriptional activities of ATAF2 using a transient assay system in this study. We used an effector consisting of a GAL4-DNA binding domain (GAL4-BD) fused to ATAF2, and observed upregulated reporter gene expression, suggesting that ATAF2 potentially has transcriptional activation activity. ATAF2 has been shown to activate reporter gene expression under the control of the ORE1 promoter. By contrast, ATAF2 significantly repressed reporter gene expression driven by the NIT2 promoter. These data suggest that ATAF2 is a bifunctional transcription factor that can alter target gene expression depending on the promoter sequences.

Long-term monitoring of bioluminescence circadian rhythms of cells in a transgenic Arabidopsis mesophyll protoplast culture

Shunji Nakamura, Tokitaka Oyama . . . . . . . 291 [PDF]

The circadian system of plants is based on the cell-autonomously oscillating circadian clock. In the plant body, these cellular clocks are associated with each other, but their basic and intrinsic properties are still largely unknown. Here we report a method that enables long-term monitoring of bioluminescence circadian rhythms of a protoplast culture in a complete synthetic medium. From the leaves of Arabidopsis transgenic plants carrying the luciferase gene under a clockgene promoter, mesophyll protoplasts were isolated and their bioluminescence was automatically measured every 20 min for more than one week. Decreasing luminescence intensities were observed in protoplasts when they were cultured in a Murashige and Skoog-based medium and also in W5 solution. This decrease was dramatically improved by adding the phytohormones auxin and cytokinin to the MS-based medium; robust circadian rhythms were successfully monitored. Interestingly, the period lengths of bioluminescence circadian rhythms of protoplasts under constant conditions were larger than those of detached leaves, suggesting that the period lengths of mesophyll cells in leaves were modulated from their intrinsic properties by the influence of other tissues/cells. The entrainability of protoplasts to light/dark signals was clearly demonstrated by using this monitoring system. By analyzing the circadian behavior of isolated protoplasts, the basic circadian system of plant cells may be better understood.

Docking analysis of models for 4-hydroxy-3-methylbut-2-enyl diphosphate reductase and a ferredoxin from Botryococcus braunii, race B

Hidenobu Uchida, Eiichi Mizohata, Shigeru Okada . . . . . . . 297 [PDF] [Supplement]

The green microalga Botryococcus braunii Showa, which produces large amounts of triterpene hydrocarbons, exclusively uses the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosyntheses, and the terminal enzyme in this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), is regarded as a light-dependent key regulatory enzyme. In order to investigate the possible association of HDR and ferredoxin in this organism, we constructed tertiary structure models of B. braunii HDR (BbHDR) and one of ferredoxin families in the alga, a photosynthetic electron transport F (BbPETF)-like protein, by using counterparts from E. coli and Chlamydomonas reinhardtii as templates, respectively, and performed docking analysis of these two proteins. After docked models are superimposed onto their counterpart proteins in a non-photosynthetic organism, Plasmodium falciparum, the BbPETF-like protein comes in contact with the backside of BbHDR, which was defined in a previous report (Rekittke et al. 2013), and the distance of the two Fe–S centers is 14.7 Å. This distance is in almost the same level as that for P. falicarum, 12.6 Å. To our knowledge, this is the first model suggesting the possible association of HDR with a ferredoxin in O2-evolving photosynthetic organisms.