Plant Biotechnology Vol.35 No.1


Original Papers

Evidence that the Arabidopsis thaliana 3-hydroxy-3-methylglutaryl-CoA reductase 1 is phosphorylated at Ser577 in planta

Jekson Robertlee, Keiko Kobayashi, Jianwei Tang, Masashi Suzuki, Toshiya Muranaka . . . . . . . 1 [PDF] [Supplement]

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is an essential enzyme in the mevalonate pathway. In higher plants, mevalonate pathway involves in the production of precursor for isoprenoids biosynthesis, including essential components for cell functions. Previously, we confirmed that the Arabidopsis thaliana HMGR1S (AtHMGR1S) is phosphorylated at S577 by the combination of sucrose non-fermenting related kinase-1 (SnRK1) and geminivirus rep-interacting kinase-1 (GRIK1) in vitro. However, even in quantitative phosphoproteomics studies that were directed to find SnRK1 target substrates, AtHMGR1S phosphorylation at S577 has never been detected in planta. In this study, we expressed AtHMGR1S as a C-terminal FLAG-fusion protein in A. thaliana hmg1 mutant to confirm its phosphorylation in planta. Our results provide the first direct evidence that AtHMGR1S is phosphorylated at S577 in planta. Moreover, phosphatase inhibitors treatment to the A. thaliana seedlings induced AtHMGR1S phosphorylation at sites other than S577, suggesting the presence of a novel HMGR regulatory mechanism in planta.

Molecular cloning of flavonoid biosynthetic genes and biochemical characterization of anthocyanin O-methyltransferase of Nemophila menziesii Hook. and Arn.

Naoko Okitsu, Takayuki Mizuno, Keisuke Matsui, Sun Hee Choi, Yoshikazu Tanaka . . . . . . . 9 [PDF] [Supplement]

Blue flower color of Nemophila menziesii Hook. and Arn. is derived from a metalloanthocyanin, nemophilin, which comprises petunidin-3-O-[6-O-(trans-p-coumaroyl)-β-glucoside]-5-O-[6-O-(malonyl)-β-glucoside], apigenin-7-O-β-glucoside-4′-O-(6-O-malonyl)-β-glucoside, and Mg2+ and Fe3+ ions. The flavonoid biosynthetic pathway of nemophilin has not yet been characterized. RNA-Seq analysis of the petals yielded 61,491 contigs. These were searched using BLAST against petunia or torenia flavonoid biosynthetic proteins, which identified 11 putative full-length protein sequences belonging to the flavonoid biosynthetic pathway. RT-PCR using primers designed on the basis of these sequences yielded 14 sequences. Spatio-temporal transcriptome analysis indicated that genes involved in the early part of the pathway are strongly expressed during early-petal development and that those in the late part at late-flower opening stages, but they are rarely expressed in leaves. Flavanone 3-hydroxylase and flavonoid 3′,5′-hydroxylase cDNAs were successfully expressed in yeast to confirm their activities. Recombinant anthocyanin O-methyltransferase cDNA (NmAMT6) produced using Escherichia coli was subjected to biochemical characterization. Km of NmAMT6 toward delphinidin 3-O-glucoside was 22 µM, which is comparable with Km values of anthocyanin O-methyltransferases from other plants. With delphinidin 3-O-glucoside as substrate, NmAMT6 almost exclusively yielded petunidin 3-O-glucoside rather than malvidin 3-O-glucoside. This specificity is consistent with the anthocyanin composition of Nemophila petals.

CLE14 peptide signaling in Arabidopsis root hair cell fate determination

Naoto Hayashi, Takuya Tetsumura, Shinichiro Sawa, Takuji Wada, Rumi Tominaga-Wada . . . . . . . 17 [PDF]

Morphological adjustment is a critical strategy for the survival of plant species in various environments. The CLE (CLAVATA3/EMBRYO SURROUNDING REGION) family of plant polypeptides is known to play important roles in various physiological and developmental processes and the relevant signaling pathways are conserved in diverse land plants. Previously, it has been suggested that overexpression of CLE14 promotes root hair cell differentiation in Arabidopsis roots. To clarify this suggested function of CLE14 peptide on root hair induction, we examined the effect of synthetic CLE14 peptide on Arabidopsis root hair development. Consistent with the results of previous overexpression analyses of CLE14, we demonstrated that application of synthetic CLE14 peptide induced excess root hair formation on CLE14-treated Arabidopsis roots. In addition, CLE14 reduced the expression of the non-hair cell fate determinant gene, GLABRA2. Our results thus indicate that CLE14 can activate the transcriptional regulatory cascade of root hair formation.

Coexpression of WUSCHEL related homeobox (WOX) 2 with WOX8 or WOX9 promotes regeneration from leaf segments and free cells in Nicotiana tabacum L.

Masaharu Kyo, Kazuna Maida, Yuki Nishioka, Koitaro Matsui . . . . . . . 23 [PDF]

To examine the effect of the ectopic expression of three Arabidopsis genes, including WOX2, WOX8 and WOX9, on the regenerative competency of tissues and cells cultured in vitro, we developed a transgenic variety of Nicotiana tabacum, in which these genes were under the transcriptional control of a chemical-inducible expression system. We designed a two-step culture method to feasibly demonstrate the effect as follows. Leaf segments of approximately 10 mm2 were prepared from transgenic plants and their hybrids and cultured in a liquid medium based on modified Murashige and Skoog medium supplemented with an auxin, 2,4-dichrorophenoxyacetic acid and/or an expression inducer β-estradiol for 10 days in dark. The segments were subsequently cultured on a solidified medium in the absence of both the auxin and inducer in light for 3 weeks. We observed remarkable regeneration of plantlets only in segments derived from the hybrids possessing two transgenes, WOX2 combined with WOX8 or WOX9, but no regeneration in the segments derived from their parental lines. We also observed that free cells released from the hybrid explants in the liquid medium developed into embryo-like structures due to the transient application of the inducer. In a wide range of species including recalcitrants, the effect of the coexpression of these genes may be useful for developing an alternative to conventional protocols that requires cytokinin.

Proteomic analysis of xylem vessel cell differentiation in VND7-inducible tobacco BY-2 cells by two-dimensional gel electrophoresis

Masahiro Noguchi, Masayuki Fujiwara, Ryosuke Sano, Yoshimi Nakano, Yoichiro Fukao, Misato Ohtani, Taku Demura . . . . . . . 31 [PDF]

The xylem vessel is an essential structure for water conduction in vascular plants. Xylem vessel cells deposit thick secondary cell walls and undergo programmed cell death, to function as water-conducting elements. Since the discovery of the plant-specific NAC domain-type VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors, which function as master switches of xylem vessel cell differentiation in Arabidopsis, much has been learned about the transcriptional regulatory network of xylem vessel cell differentiation. However, little is known about proteome dynamics during xylem vessel cell differentiation. Here, we performed two-dimensional electrophoresis-based proteomic analysis of xylem vessel cell differentiation using a transgenic tobacco BY-2 cell line carrying the VND7-inducible system (BY-2/35S::VND7-VP16-GR), in which synchronous trans-differentiation into xylem vessel cells can be induced by the application of a glucocorticoid. Of the 47 spots revealed by gel electrophoresis, we successfully identified 40 proteins. Seventeen proteins, including several well-characterized proteins such as a cysteine protease and serine carboxypeptidase (involved in programmed cell death), were upregulated after 24 h of induction. However, previous transcriptomic analysis showed that only eight of these proteins are upregulated at the transcriptional level during xylem vessel cell differentiation in BY-2/35S::VND7-VP16-GR cells. These findings suggest that post-transcriptional regulation strongly affects proteomic dynamics during xylem vessel cell differentiation.

A genetic link between epigenetic repressor AS1–AS2 and DNA replication factors in establishment of adaxial–abaxial leaf polarity of Arabidopsis

Toan Quy Luong, Sumie Keta, Toshiharu Asai, Shoko Kojima, Ayami Nakagawa, José Luis Micol, Shitou Xia, Yasunori Machida, Chiyoko Machida . . . . . . . 39 [PDF] [Supplement]

Balanced development of adaxial and abaxial domains in leaf primordia is critical for the formation of flat symmetric leaf lamina. Arabidopsis ASYMMETRIC LEAVES1 (AS1) and AS2 proteins form a complex (AS1–AS2), which acts as key regulators for the adaxial development by the direct repression of expression of the abaxial gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). Many modifier mutations have been identified, which enhance the defect of as1 and as2 mutations to generate abaxialized filamentous leaves without adaxial traits, suggesting that the development of the adaxial domain is achieved by cooperative repression by AS1–AS2 and the wild-type proteins corresponding to the modifiers. Mutations of several genes for DNA replication-related chromatin remodeling factors such as Chromatin Assembly Factor-1 (CAF-1) have been also identified as modifiers. It is still unknown, however, whether mutations in genes involved in DNA replication themselves might act as modifiers. Here we report that as1 and as2 mutants grown in the presence of hydroxyurea, a known inhibitor of DNA replication, form abaxialized filamentous leaves in a concentration-dependent manner. We further show that a mutation of the INCURVATA2 (ICU2) gene, which encodes the putative catalytic subunit of DNA polymerase α, and a mutation of the Replication Factor C Subunit3 (RFC3) gene, which encodes a protein used in replication as a clamp loader, act as modifiers. In addition, as2-1 icu2-1 double mutants showed increased mRNA levels of the genes for leaf abaxialization. These results suggest a tight link between DNA replication and the function of AS1–AS2 in the development of flat leaves.

Development of an efficient regeneration system for the precious and fast-growing timber tree Toona ciliata

Pei Li, Yuanyuan Shang, Wei Zhou, Xinsheng Hu, Wenmai Mao, Jingjian Li, Juncheng Li, Xiaoyang Chen . . . . . . . 51 [PDF]

Toona ciliata (Chinese mahogany) is an important timber species and secondary protected plant due to excessive exploitation in China. Here we developed a robust and efficient regeneration system for adventitious shoot induction using hypocotyl explants of T. ciliata. To facilitate plant growth, different regulators were added to Murashige–Skoog (MS) medium (0.5 mg/l 6-BA, 1.0 mg/l KT and 0.1 mg/l IBA). A regeneration frequency of 58.67% with four shoots per explant was achieved by horizontal setting of hypocotyls on MS medium and following a 20-day seeding period. MS medium supplemented with 0.3 mg/l 6-BA and 0.2 mg/l NAA was optimal for shoot multiplication and elongation, with a multiplication coefficient of 3.06. A rooting frequency of 93.33% was achieved using the half-strength MS containing 0.1 mg/l NAA. After acclimatization, plantlets were transplanted to sterilized nutrient soil containing a 2 : 1 ratio of vermiculate with 90% survival frequency. Thus, the regeneration system developed in this study would be useful for genetic transformation and other biotechnology endeavours in T. ciliata.

Identification and characterization of two ammonium transporter genes in flowering Chinese cabbage (Brassica campestris)

Yunna Zhu, Yanwei Hao, Houcheng Liu, Guangwen Sun, Riyuan Chen, Shiwei Song . . . . . . . 59 [PDF]

Ammonium transporters (AMTs), which include AMT1 and AMT2 subfamilies, have been identified and partially characterized in many plants. In this study, two AMT2-type genes from Brassica campestris, namely BcAMT2 and BcAMT2like, were identified and characterized. BcAMT2 and BcAMT2like are 2666 bp and 2952 bp, encode proteins of 490 and 489 amino acids, respectively, and contain five exons and four introns. Transient expression of these proteins labelled with green fluorescence protein in onion epidermal cells indicated that both are located on the plasma membrane. When expressing BcAMT2 or BcAMT2like, the mutant yeast strain 31019b could grow on medium containing 2 mM ammonium as the only nitrogen source when expressing BcAMT2 or BcAMT2like, indicating that both are functional AMT genes. Quantitative PCR results showed that BcAMT2 and BcAMT2like were expressed in all tissues, but they displayed different expression patterns in the reproductive stages. BcAMT2s transcript levels in leaves were positively correlated with ammonium concentration and external pH. Moreover, the expression BcAMT2s responded to diurnal change. Furthermore, the uncharged form of ammonium, i.e., ammonia, might also be transported by BcAMT2s. These results provide new insights into the molecular mechanisms underlying ammonium absorption and transportation by the AMT2 subfamily in B. campestris.

Expression of barley Glutathione S-Transferase13 gene reduces accumulation of reactive oxygen species by trichothecenes and paraquat in Arabidopsis plants

Ninik Nihayatul Wahibah, Tomokazu Tsutsui, Daisuke Tamaoki, Kazuhiro Sato, Takumi Nishiuchi . . . . . . . 71 [PDF] [Supplement]

Glutathione S-transferases (GSTs) play an important role in the detoxification of reactive oxygen species (ROS) and toxic compounds. We found that the barley phi class GST (HvGST13) gene is upregulated by trichothecene phytotoxin produced by the fungal pathogen Fusarium graminearum in barley. Trichothecene phytotoxins such as DON and T-2 toxin induce accumulation of ROS and cell death in plants. It is known that the death of host cells contributes to the virulence of F. graminearum during the later stages of infection. To characterize the role of the HvGST13 gene, we generated Arabidopsis plants in which HvGST13 was overexpressed. Growth inhibition by DON and T-2 toxin was significantly alleviated in the HvGST13ox Arabidopsis plants compared with the wild type. Accumulation of ROS and cell death apparently decreased in HvGST13ox Arabidopsis plants treated with trichothecene. Paraquat herbicide is well known to induce the generation of ROS in plants. Paraquat-induced growth retardation was also suppressed in the HvGST13ox Arabidopsis plants compared with wild type. The inoculation of F. graminearum causes disease symptoms that are markedly decreased in HvGST13ox Arabidopsis plants compared to those in the wild type. Therefore, the HvGST13 gene suppressed the phytotoxic activity of trichothecenes in plants, possibly by the scavenging of ROS.

Short Communication

Induction, identification and characterization of polyploidy in Stevia rebaudiana Bertoni

Hong Zhang, Shaoya An, Juan Hu, Zhe Lin, Xiang Liu, Han Bao, Ren Chen . . . . . . . 81 [PDF]

Stevia rebaudiana Bertoni produces sweet steviol glycosides; these are regarded as the most promising substitute for sucrose and have a wide range of applications with high economic value. To improve steviol glycosides yields, we established an efficient method for inducing polyploidy in S. rebaudiana. Treatment with 0.05% colchicine for 48 h or with 0.1% colchicine for 24 h efficiently induced polyploidy in germinating seeds and polyploidy could be accurately identified using flow cytometry. Most of the tetraploid S. rebaudiana plants exhibited apparent variations in cytological and morphological characteristics, and had higher contents of the two main steviol glycosides, stevioside and rebaudioside A, than the diploid controls. These tetraploid plants may be further selected for breeding purposes or micropropagated for commercial production.


The function of ETHYLENE RESPONSE FACTOR genes in the light-induced anthocyanin production of Arabidopsis thaliana leaves

Tomotsugu Koyama, Fumihiko Sato . . . . . . . 87 [PDF]

Plants grow under threats of environmental changes that could injure cellular viability and damage whole-plant physiology. To defend themselves against such threats, plants induce protective responses, including the production of defense molecules. The red/purple pigment anthocyanin is synthesized upon leaf and fruit development as well as environmental stimuli such as excess light exposure. Therefore, the anthocyanin biosynthesis is considered as a model signaling pathway of the integration of developmental and environmental responses. This integration is tightly regulated by transcription factors, but the integrative mode of these signaling pathways has received little attention. In this study, using an Arabidopsis mutant with mutation in two ETHYLENE RESPONSE FACTOR (ERF) genes, AtERF4 and AtERF8, we investigated the regulatory signaling pathway that leads to the production of anthocyanin in response to light. We detected the accumulation of anthocyanin in detached leaves after incubation on water under light illumination and intact leaves after being transferred into the strong light condition, suggesting that the photoinhibition mediated the production of anthocyanin. Our results demonstrated that the erf mutant decreased the rate and extent of the production of anthocyanin in association with changes of the transcript levels of anthocyanin-biosynthetic genes. As these ERF genes are known regulators of leaf senescence—the final stage of leaf development—we provide an insight into the ERF-mediated integration of two regulatory pathways of the light response and developmental age.