—Invited Paper—

AgarTrap Protocols on your Benchtop: Simple Methods for Agrobacterium-mediated Genetic Transformation of the Liverwort Marchantia polymorpha

Shoko Tsuboyama, Yutaka Kodama . . . . . . . 93[PDF]

Agrobacterium-mediated genetic transformation is a powerful technique in plant biology. We recently developed a simplified Agrobacterium-mediated genetic transformation method for the liverwort Marchantia polymorpha, named AgarTrap (agar-utilized transformation with pouring solutions). AgarTrap is easy to perform; all procedures can be completed within a week using a single plate of solid medium, and basic operations involve simply pouring the appropriate solutions onto the solid medium. Thus far, we have developed three types of AgarTrap methods (S-AgarTrap, G-AgarTrap, and T-AgarTrap) using three different M. polymorpha tissues: sporelings, intact gemmalings, and mature thallus pieces, respectively. Each AgarTrap method can be used to transform tissues at high efficiency, thereby producing sufficient numbers of transformants for study. The ease and efficiency of these AgarTrap methods will likely prompt widespread molecular biological analyses of M. polymorpha. In this review, we describe the basic characteristics of the three AgarTrap methods and present the detailed protocols used in our laboratory.

Original Papers

Characterization of rice KT/HAK/KUP potassium transporters and K+ uptake by HAK1 from Oryza sativa

Tomoyuki Okada, Sousuke Yamane, Masatoshi Yamaguchi, Ko Kato, Atsuhiko Shinmyo, Yuta Tsunemitsu, Kozo Iwasaki, Daisei Ueno, Taku Demura . . . . . . . 101[PDF] [Supplement]

Plant high-affinity K+ (HAK) transporters are divided into four major clusters. Cluster I transporters, in particular, are thought to have high-affinity for K+. Of the 27 HAK genes in rice, eight HAK transporters belong to cluster
I. In this study, we investigated the temporal expression patterns during K+ deficiency and K+ transport activity of these eight HAK transporters. The expression of seven HAK genes except OsHAK20 was detected. Expression of OsHAK1, OsHAK5 and OsHAK21 was induced in response to K+ deficiency; however, that of other genes was not. Six of the eight HAK transporters—OsHAK1, OsHAK5, OsHAK19, OsHAK20, OsHAK21, and OsHAK27—complemented the K+– transporter-deficient yeast or bacterial strain. Further, the yeast cells expressing OsHAK1 were more sensitive to Na+ than those expressing OsHAK5. Mutant analysis showed that the high-affinity K+ uptake activity was almost undetectable in oshak1 mutants in a low-K+ medium (0.02 mM). In addition, the high-affinity K+ uptake activity of wild-type plants was inhibited by mild salt stress (20 mM NaCl); however, Na+ permeability of OsHAK1 was not detected in Escherichia coli cells. The high-affinity K+ uptake activity by leaf blades was detected in wild-type plants, while it was not detected in oshak1 mutants. Our results suggest that OsHAK1 and OsHAK5 are the two important components of cluster I corresponding to low-K+ conditions, and that the transport activity of OsHAK1, unlike that of OsHAK5, is sensitive to Na+. Further, OsHAK1 is suggested to involve in foliar K+ uptake.

Identification and characterization of a novel sesquiterpene synthase, 4-amorphen-11-ol synthase, from Artemisia maritima

Paskorn Muangphrom, Hikaru Seki, Seiya Matsumoto, Mika Nishiwaki, Ery O. Fukushima, Toshiya Muranaka . . . . . . . 113[PDF] [Supplement]

Artemisinin, a sesquiterpene lactone exhibiting effective antimalarial activity, is produced by only Artemisia annua plant. A key step in artemisinin biosynthesis is the cyclization of farnesyl pyrophosphate (FPP) to amorpha-4,11- diene catalyzed by amorpha-4,11-diene synthase (AaADS). Intriguingly, several non-artemisinin-producing Artemisia plants also express genes highly homologous to AaADS. Our previous functional analysis of these homologous enzymes revealed that they catalyzed the synthesis of rare natural sesquiterpenoids. In this study, we analyzed the function of another putative sesquiterpene synthase highly homologous to AaADS from A. maritima. Unlike AaADS, in vivo enzymatic assay showed that this enzyme cyclized FPP to 4-amorphen-11-ol, a precursor of several gastroprotective agents. The discovery of 4-amorphen-11-ol synthase (AmAOS) and the successful de novo production of 4-amorphen-11-ol in engineered yeast demonstrated herein provides insights into the methods used to enhance its production for future application.

Isolation and characterization of rhizobia from nodules of Clitoria ternatea in Thailand

Mallika Duangkhet, Yamikani Chikoti, Apiraya Thepsukhon, Pilunthana Thapanapongworakul, Sirinapa Chungopast, Shigeyuki Tajima, Mika Nomura . . . . . . . 123[PDF]

Rhizobia were isolated from the root nodules of Clitoria ternatea in Thailand. The phylogeny of the isolates was investigated using 16S rDNA and the internal transcribed spacer (ITS) region from 16S to 23S rDNA. The phylogenetic tree of the 16S rDNA showed that ten of the eleven isolates belonged to Bradyrhizobium elkanii, and one belonged to Bradyrhizobium japonicum. The topology of the ITS tree was similar to that of 16S rDNA. The acetylene reduction activity was higher for the nodules inoculated with the isolated B. elkanii strains than for those inoculated with B. japonicum strains. When C. ternatea plants were inoculated with various Bradyrhizobium USDA strains isolated from Glycine max, C. ternatea formed many effective nodules with B. elkanii, especially USDA61. However, acetylene reduction activity per plant and the growth were higher in C. ternatea inoculated with our isolates. From these data we propose that effective rhizobia inoculant were identified for C. ternatea cultivation.

Comparative analysis of CYP716A subfamily enzymes for the heterologous production of C-28 oxidized triterpenoids in transgenic yeast

Hayato Suzuki, Ery Odette Fukushima, Naoyuki Umemoto, Kiyoshi Ohyama, Hikaru Seki, Toshiya Muranaka . . . . . . . 131[PDF] [Supplement1] [Supplement2] [Supplement3] [Supplement4]

Several enzymes of the CYP716A subfamily have been reported to be involved in triterpenoid biosynthesis. Members of this subfamily oxidize various positions along the triterpenoid backbone and the majority of them catalyze a three-step oxidation at the C-28 position. Interestingly, C-28 oxidation is a common feature in oleanolic acid, ursolic acid, and betulinic acid, which are widely distributed in plants and exhibit important biological activities. In this work, three additional CYP716A enzymes isolated from olive, sugar beet, and coffee, were characterized as multifunctional C-28 oxidases. Semi-quantitative comparisons of in vivo catalytic activity were made against the previously characterized enzymes CYP716A12, CYP716A15, and CYP716A52v2. When heterologously expressed in yeast, the isolated enzymes differed in both catalytic activity and substrate specificity. This study indicates that the screening of enzymes from different plants could be a useful means of identifying enzymes with enhanced catalytic activity and desired substrate specificity. Furthermore, we show that “naturally-evolved” enzymes can be useful in the heterologous production of pharmacologically and industrially important triterpenoids.

The functional balance between the WUSCHEL-RELATED HOMEOBOX1 gene and the phytohormone auxin is a key factor for cell proliferation in Arabidopsis seedlings

Miyuki T. Nakata, Toshiaki Tameshige, Masahiro Takahara, Nobutaka Mitsuda, Kiyotaka Okada . . . . . . . 141[PDF] [Supplement]

The WUSCHEL-RELATED HOMEOBOX1 (WOX1) transcription factor and its homolog PRESSED FLOWER (PRS) are multifunctional regulators of leaf development that act as transcriptional repressors. These genes promote cell proliferation under certain conditions, but the related molecular mechanisms are not well understood. Here, we present a new function for WOX1 in cell proliferation. To identify the WOX1 downstream genes, we performed a microarray analysis of shoot apices of transgenic Arabidopsis thaliana lines harboring [35Sp::WOX1-glucocorticoid receptor (GR)] in which the WOX1 function was temporarily enhanced by dexamethasone. The downregulated genes were significantly enriched for the Gene Ontology term “response to auxin stimulus”, whereas the significantly upregulated genes contained auxin transport-associated PIN1 and AUX1 and the auxin response factor MP, which are involved in formation of auxin response maxima. Simultaneous treatments of synthetic auxin and dexamethasone induced the formation of green compact calli and the unorganized proliferation of cells in the hypocotyl. A microarray analysis of 35Sp::WOX1-GR plants treated with indole-3-acetic acid and dexamethasone revealed that WOX1 and auxin additively influenced their common downstream genes. Furthermore, in the presence of an auxin-transport inhibitor, cell proliferation during leaf initiation was suppressed in the prs mutant but induced in a broad region of the peripheral zone of the shoot apical meristem in the ectopic WOX1– expressing line FILp::WOX1. Thus, our results clarify the additive effect of WOX1/PRS and auxin on their common downstream genes and highlight the importance of the balance between their functions in controlling cell proliferation.

Short Communications

—Invited Paper—

ARF GTPase machinery at the plasma membrane regulates auxin transport-mediated plant growth

Satoshi Naramoto, Junko Kyozuka . . . . . . . 155[PDF]

VAN3 is a plant ACAP-type ADP-ribosylation factor-GTPase activating protein (ARF-GAP) that regulates auxin transport-mediated plant morphogenesis such as continuous venation and lateral root development in Arabidopsis. Previous studies suggested that VAN3 localizes at the plasma membrane (PM) and intracellular structures. However, the role of PM localization in mediating the van3 mutant phenotype is not clear. Here we performed subcellular localization analysis of VAN3 and its regulators CVP2 and VAB to determine their endogenous functions. We found that GFP-tagged CVP2 and VAB preferentially localize at the PM in stably transformed plants. We determined that transgenic plants with lower expression levels of GFP- or mRFP-tagged VAN3 displayed PM localization, which was sufficient to rescue the van3 mutant. Functional VAN3-mRFP and VAB-GFP colocalized at PMs. The van3 mutant phenotype was suppressed by mutation of VAN7/GNOM, which encodes an ARF-GEF that localizes at the PM and Golgi apparatus. These combined results suggest that ARF-GTPase machinery at the PM regulates auxin transport-mediated plant growth and development.

An improved anther culture procedure for obtaining new commercial Mediterranean temperate japonica rice (Oryza sativa) genotypes

Camilo López-Cristoffanini, Xavier Serrat, Eduardo Ramos-Fuentes, Isidre Hooghvorst, Roser Llaó, Marta López-Carbonell, Salvador Nogués . . . . . . . 161[PDF]

Rice is one of the greatest calorie supply for the world population, especially since its production is almost entirely destined to direct human consumption and its demand will increase along with the world population. There are efforts worldwide to increase rice yields by obtaining new improved and stabilized rice lines. The rice anther culture, a fast and cheap technique, allows to obtain double haploid lines in less than one year. We report its application with an improved protocol in four Mediterranean japonica rice genotypes at F2 generation. We performed a screening test for cold-pretreatment at 5.0±0.1℃ and concluded that the optimum duration was 9 days as it produced the higher rate of anther-derived callus induction. This revised protocol was successfully applied to the four genotypes, obtaining good results in all the procedure’s steps. At the end, more than 100 of double haploid green plants were generated. Moreover, 9 lines obtained from the anther culture procedure showed good qualities for the Spanish market at the growing, farming and grain production level during the field assays. Therefore, we report an improved anther culture procedure for obtaining double haploid lines from temperate japonica rice genotypes showing high commercialization expectance.

Sugar-responsive transcription factor bZIP3 affects leaf shape in Arabidopsis plants

Miho Sanagi, Yu Lu, Shoki Aoyama, Yoshie Morita, Nobutaka Mitsuda, Miho Ikeda, Masaru Ohme-Takagi, Takeo Sato, Junji Yamaguchi . . . . . . . 167[PDF] [Supplement]

Sugars are essential for plant metabolism, growth and development. Plants must therefore manage their growth and developmental processes in response to sugar availability. Sugar signaling pathways constitute a complicated molecular network and are associated with global transcriptional regulation. However, the molecular mechanisms underlying sugar signaling remain largely unclear. This study reports that the protein basic-region leucine zipper 3 (bZIP3) is a novel sugar- responsive transcription factor in Arabidopsis plants. The expression of bZIP3 was rapidly repressed by sugar. Genetic analysis indicated that bZIP3 expression was modulated by the SNF1-RELATED KINASE 1 (SnRK1) pathway. Moreover, transgenic plants overexpressing bZIP3 and dominant repressor form bZIP3-SRDX showed aberrant shaped cotyledons with hyponastic bending. These findings suggest that bZIP3 plays a role in plant responses to sugars and is also associated with leaf development.


Control of plant growth and development by overexpressing MAP3K17, an ABA-inducible MAP3K, in Arabidopsis

Daisuke Matsuoka, Kaori Soga, Takuto Yasufuku, Takashi Nanmori . . . . . . . 171[PDF]

Abscisic acid (ABA) plays an important role in plant growth, development, and stress responses. ABA regulates many aspects of plant growth and development, including seed maturation, dormancy, germination, the transition from vegetative to reproductive growth, leaf senescence and responses to environmental stresses, such as drought, high salinity and cold. It is also known that mitogen-activated protein kinase (MAPK) cascades function in ABA signaling. Recently, we and another group have identified the ABA-inducible MAP3Ks MAP3K17 and MAP3K18 as the upstream MAP3Ks of MKK3, implicating the MAP3K17/18-MKK3-MPK1/2/7/14 cascade in ABA signaling. It has also been reported that overexpression of MAP3K18 in Arabidopsis causes an early leaf senescence phenotype, ABA hypersensitive stomata closing, and drought tolerance. In this study, we generated transgenic plants overexpressing MAP3K17 (35S:MAP3K17) and its kinase-inactive form (35S:MAP3K17KN). The bolting of 35S:MAP3K17 was earlier than WT, and the fresh weights of the seedlings were smaller, whereas 35S:MAP3K17KN showed the opposite phenotype. These results indicate that the transition from vegetative to reproductive growth can be regulated by overexpression of MAP3K17 and its kinase-inactive form. Moreover, 35S:MAP3K17 showed lower sensitivity to ABA during post-germinated growth, whereas 35S:MAP3K17 KN showed the opposite phenotype, suggesting the negative roles of MAP3K17 in the response to ABA. Our work provides the possibility to regulate plant growth and development by the genetic manipulation of ABA-induced MAPK cascades, leading to improved crop growth and productivity.

Efficient identification of NLR by using a genome-wide protein domain and motif survey program, Ex-DOMAIN

Mari Narusaka, Harunobu Yunokawa, Yoshihiro Narusaka . . . . . . . 177[PDF] [Supplement1] [Supplement2]

Genomic and amino acid sequences of organisms are freely available from various public databases. We designed a genome-wide survey program, named “Ex-DOMAIN” (exhaustive domain and motif annotator using InterProScan), of protein domains and motifs to aid in the identification and characterization of proteins by using the InterProScan sequence analysis application, which can display information and annotations of targeted proteins and genes, conserved protein domains and motifs, chromosomal locations, and structural diversities of target proteins. In this study, we indicated the disease resistance genes (proteins) that play an important role in defense against pathogens in Arabidopsis thaliana (thale cress) and Cucumis sativus (cucumber), by searches based on the conserved protein domains, NB-ARC (a nucleotide- binding adaptor shared by the apoptotic protease-activating factor-1, plant resistance proteins, and Caenorhabditis elegans death-4 protein) and C-terminal leucine-rich repeat (LRR), in the nucleotide-binding domain and LRR (NLR) proteins. Our findings suggest that this program will enable searches for various protein domains and motifs in all organisms.

Development of a stable Agrobacterium-mediated transformation protocol for Sorghum bicolor Tx430

Kanna Sato-Izawa, Kyoko Tokue, Hiroshi Ezura . . . . . . . 181[PDF] [Supplement]

Sorghum is a recalcitrant crop for Agrobacterium-mediated genetic transformation. Several parameters related to Agrobacterium-mediated transformation were tested to optimize sorghum transformation frequencies. In this study, we evaluated pretreatment of sorghum variety Tx430 immature embryos using Agrobacterium strain GV2260. Pretreatment of immature embryos with heat (43℃) treatment for 15 or 21 min, and centrifugation resulted in a transformation efficiency of up to 1.9% of immature embryos treated. Although further optimization to enhance transformation efficiency is required, this study contributes to the genetic validation of genes of interest and molecular breeding in sorghum plants.