Thioglucosid glucohydrolase (myrosinase), TGG1, is a strikingly sbundant protein in Arabidopsis guard cells. We investigated responses of tgg1-3, tgg2-1 and tgg1-3 tgg2-1 double mutants to ABA and MeJA to clarify whether two myrosinases, TGG1 and TGG2, function during stomatal closure. ABA, MeJA and H₂O₂ induced stomatal closure in wild ype, tgg1-3 and tgg2-1, but failed to induce stomatal closure in tgg1-3 tgg2-1. All mutants and wild type showed Ca²⁺-induced stomatal closure and ABA-induced reactive oxygen species (ROS) production. A model is discussed in which two myrosinases redundantrly function downstream of ROS production and upstream of cytosolic Ca²⁺ elevation in ABA and MeJA signaling in guard cells.

View full abstract

Methyl jasmonate (MeJA) induces stomatal closure dependent on change of cytosolic Ca2+ concentration in guard cells. However, details of this molecular mechanism remain unclear. Calcium-dependent protein kinases (CDPKs) function as Ca2+ signal transducers in various plant physiological processes. It was suggested that Arabidopsis four CDPKs, CPK3, CPK6, CPK4, and CPK11 function as positive regulators in abscisic acid signaling in guard cells. Here, we examined roles of these CDPKs in MeJA signaling in guard cells using each CDPK knockout mutant. In CPK6 gene disruption mutants, MeJA-induced stomatal closure was impaired whereas in CPK3, CPK4, and CPK11 gene disruption mutants, MeJA-induced stomatal closure were not altered. We also evaluated roles of CPK6 in MeJA regulation of second messenger production and ion channel activity in guard cells. Our results provide genetic evidence that CPK6 functions as a positive regulator of MeJA signaling in Arabidopsis guard cells.

View full abstract

We have demonstrated that cryptochromes (cry) mediate blue light dependent photomorphogenetic responses in rice. But it remains unclear how blue light signals are transduced in rice.
To identify blue light signaling factors, we screened O. glumaepatula / Taichu-65 introgression lines, which were prepared by Kyushu University, for different phenotypes grown under blue light. Among them, IL113 showed deeper declination of second leaf under blue light than WT did, while Red, Far-red or White light did not induce any differences in phenotypes between IL113 and WT. Moreover, lengths of leaf sheaths in IL113 were as long as those in WT even under blue light. Therefore, it was suggested that IL113 was hyper sensitive to blue light for the leaf declination. We speculated that the substitution chromosomal segment on the long arm of chromosome 4 is related with blue light dependent declination of leaf, because any lines didn't exhibit deeper declination of leaf under blue light.
We analyzed the growing process of rice seedlings grown under blue light in IL113 and Taichu-65, using original monitoring system. We will report the growing process of these lines under blue light

View full abstract

Stomata open in response to a weak blue light under a background of strong red light. Blue light activates the plasma membrane H⁺-ATPase and induces hyperpolarization of the plasma membrane. The hyperpolarization drives K⁺ uptake through voltage-gated K⁺ channel, and the accumulated K⁺ is electrically compensated by malate formed in guard cells. Red light drives photosynthetic electron transport in guard-cell chloroplasts, and is suggested to provide ATP for H⁺-ATPase and reductant for malate formation in the cytosol. Here, we investigated in malate formation and stomatal opening under various light conditions. We confirmed the synergistic effect of red and blue light on malate accumulation and stomatal opening, and found that both responses were inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, a PSII inhibitor. The responses were also inhibited by vanadate, a H⁺-ATPase inhibitor. These results suggest that photosynthetic electron transport in guard-cell chloroplasts is responsible for the malate formation and stomatal opening by blue light.

View full abstract

Leaf peroxisomes participate in photorespiration with mitochondria and chloroplasts. Here, we have studied the interaction between peroxisomes with mitochondria and chloroplasts to elucidate the role of the interaction in metabolic pathway. The results showed that the interaction of peroxisomes with chloroplasts is enhanced under light condition with elongation of peroxisomes along chloroplasts. Further analyses with two-photon laser scanning microscopy showed that the interaction is regulated by blue and red light. Then, we investigated the effect of photosynthetic inhibitors, DCMU and DBMIB, on the interaction of peroxisomes with mitochondria and chloroplasts. The results showed that peroxisomes and mitochondria are both sphere and weaken the interaction with chloroplasts similar to that in dark condition. The interaction of peroxisomes with chloroplasts was lost in white sector of var2, indicating that the interaction between these organelles is regulated by photosynthesis. These findings suggest that the photosynthetic regulation of the interaction between these three organelles would be reasonable for efficient metabolite flow in photorespiration.

View full abstract

In the yellow and colorless mutant of Chlorella kesseleri, grown with nitrate as the sole nitrogen source, the addition of glucose or the irradiation of blue light inhibited the uptakes of nitrate, amino acids and ammonia in growing cells, while these enhanced the uptakes of nitrate, amino acids and ammonia in starved cells. For the colorless mutant, blue light was shown to activate purified nitrate reductase and also activate glycine oxidase. It is known that Chlorella kesseleri, grown with ammonia as the sole nitrogen source has no nitrate reductase protein.
In this experiment, we observed that ammonia-grown cells showed blue light-induced respiration in similar manner to nitrate-grown cells. These results indicate that in the mutant cells of Chlorella examined, uptake system of nitrogenous compounds such as nitrate, amino acids and ammonia seems to be influenced by two blue light-receptors mediating the effects of ammonia accumulated in the presence of glucose or under blue light.

View full abstract

VOZ (Vascular plant One Zinc-finger protein) family is a one-zinc finger type DNA binding protein, which is highly conserved among land plants. Genetic analyses on two Arabidopsis VOZs(AtVOZ1/2) have revealed that AtVOZs are involved in flowering and cold response. However, details of AtVOZs as transcription factor remain largely unclear. In this work, we report that AtVOZs specifically interact with a transcriptional co-activator MBF1 (Multiprotein Binding Factor 1). Out of three Arabidopsis MBF1s (AtMBF1a,b,c), only AtMBF1b interacted with AtVOZ1 and AtVOZ2 in Y2H and in vitro pull down assay. Promoter-GUS reporter analysis revealed that AtVOZs and AtMBF1b co-expressed in vascular tissues. Furthermore, taken together with flowering time and freezing tolerance phenotypes in AtVOZs and AtMBF1b mutants, we will discuss molecular roles of AtVOZs and AtMBF1b in transcriptional regulation.

View full abstract

It is known that translation from most of mRNA is inhibited in plants exposed to abiotic stresses (translationally inhibited). However, a subset of mRNA maintains the translational level under these stress conditions (preferentially translated). Previously, we have identified mRNAs which maintain translation under heat stress (HS) in Arabidopsis thaliana T87 cells. It is thought that 5'UTRs have an important role in translational regulation under abiotic stress. Therefore, in this work, the importance of 5'UTRs on translational regulation has been examined in detail. At first, two expression vectors that include a GUS gene with At1g77120 5'UTR (preferentially translated) or At3g47610 5'UTR (translationally inhibited) were constructed. The generated transformants were incubated under normal or HS condition, and the translational state of each chimeric GUS mRNA was analyzed. As a result, they showed the same profile as their original mRNAs. This result indicates the importance of 5'UTR as the determinant of a translational state under HS. The sequence motifs of 5'UTR involved in translational regulation under HS are presented as well.

View full abstract

Phosphatidylcholine (PC) turnover is known as an adaptive response of eukaryotic cell to abiotic stress. In plants, PC is the major phospholipid constitutes of cell membrane and synthesized via CDP-choline pathway. The first step is catalyzed by choline kinase (CK). We obtained Arabidopsis T-DNA insertion lines of CK isogenes, and found that ck2 (At1g74320) mutant showed late-flowering. Under control condition, PC content in leaves of ck2 mutant was decreased about 18% compared to WT. Germination rate of ck2 and ck3 (At4g09760) mutants in medium supplemented with NaCl were decreased compared to WT. When seedlings were grown with NaCl after germination, ck2 and ck3 mutant showed significantly decreased biomass production. In Arabidopsis, CK2 is thought to be involved in PC production of leaves and plant growth, and the function of CK3 is important for maintaining NaCl tolerance of plants. Now, we investigate the CK activity in ck k/o and antisense-knockdown plants under salt stress.

View full abstract

UVB can damage DNA by causing formation of cyclobutane pyrimidine dimer (CPD). Photoreactivation mediated by enzyme, photolyase, is the major pathway for repairing CPD in plant, and CPD photolyase is a crucial factor for determining UVB sensitivity in plants. We previously reported that CPD photolyase functions in nuclei, chloroplasts and mitochondria in rice. This result indicates that CPD photolyase, which is encoded by a single-copy gene in the nuclear genome, translocates to chloroplasts, mitochondria, and nuclei. Thus, CPD photolyase is subjected to "triple targeting" in rice cell. However, organelle-targeting sequence of CPD photolyase has not been identified. In this study, in order to the identify the organelle-targeting amino acid sequence, we constructed expression vectors encoding protein chimeras of GFP and C-terminal (amino acids 385-506) rice CPD photolyase. The expression vector was introduced into guard cell of Vicia faba or epidermal cell of onion, and cells were examined by fluorescence microscopy. These analyses showed that the C-terminus of rice CPD photolyase contains a functional targeting sequence for transport into nuclei and mitochondria.

View full abstract

Cyclobutane pyrimidine dimer (CPD) is a major type of UV-induced DNA damage. CPD photolyase is a crucial factor for determining the sensitivity of rice to UVB. We previously reported that the native rice CPD photolyase was phosphorylated. To investigate the effect of phosphorylation on CPD photolyase function, we tried to determine the phosphorylation site of rice CPD photolyase using site-directed mutagenesis method. We previously reported that the E. coli-expressed rice CPD photolyase was not phosphorylated. Thus, we used an insect cell-free translation system, which has eukaryotic posttranslational modification machinery. We found that rice CPD photolyase, which was synthesized using insect cell-free translation system, was phosphorylated. Then, we constructed plasmids carrying the genes encoding rice CPD photolyase which replaced the putative phosphorylation site with alanine residue by site-directed mutagenesis method. The point-mutated proteins were synthesized using the plasmids by an insect cell-free translation system. In this study, we discuss about the effects of phosphorylation of CPD photolyase on its function.

View full abstract

Cyanidioschyzon merolae is unicellular photosynthetic eukaryote that inhabits in high temperature (42^oC) and acid (pH2.5) conditions. The aim of this study is to produce transgenic stresstolerant Arabidopsis thaliana by expressing genes that are involved in adaptation of C. merolae to extreme environment. Based on EST analysis of C. merolae, we found that stromal ascorbate peroxidase (stAPX) that scavenges ROS was expressed at high level (4th of 4479 entries). To examine effect of overexpression stAPX, C. merolae stAPX (CmstAPX) or A. thaliana stAPX (AtstAPX) was stably overexpressed in A. thaliana and analyzed. Immunofluorescence microscopy showed that CmstAPX was localized in the stroma of chloroplasts in A. thaliana. CmstAPX-ox plants showed higher activities of soluble APX than those of wild-type and AtstAPX-ox plants. CmstAPX-ox plants were more tolerant to oxidative stress induced by methylviologen and high-temperature stress¹. In this meeting, we will report the results of detailed analysis.
¹Hirooka S et al. Expression of the Cyanidioschyzon merolae stromal ascorbate peroxidase in Arabidopsis thaliana enhances thermotolerance. (2009) Plant Cell Rep. Dec;28:1881-1893.

View full abstract

Nucleolin is involved in various steps of ribosome synthesis such as transcription and processing of pre-rRNA and assembly with ribosomal proteins. Arabidopsis thaliana contains two genes for nucleolin; AtNuc-L1 expressed in young tissues and AtNuc-L2 expressed only weakly in floral buds under normal conditions. Expression of AtNuc-L1 is inducible by sugars and linked with cell proliferation, and its knockout mutant (atnuc1-1) shows delayed growth and various developmental defects. Although knockout of AtNuc-L2 does not affect growth, expression of AtNuc-L2 is high in atnuc1-1 and double knockout of both genes is embryonic lethal. When the wild type plants were transferred from 22^oC to 15^oC, AtNuc-L1 mRNA was induced within 1 day and the level of AtNuc-L1 protein also increased. The atnuc1-1 plants stopped growth at 15^oC. Expression of AtNuc-L1 under the RPS5A promoter in atnuc1-1 caused 20 to 70% recovery of AtNuc-L1 mRNA, and resulted in partial recovery from various developmental defects and growth arrest at 15^oC. The reduced levels of 25S and 18S rRNAs also showed partial recovery. Thus, AtNuc-L1 is necessary for growth under low temperature through ribosome synthesis.

View full abstract

LEA proteins are highly hydrophilic with random structures and boiling-soluble characteristic. LEA proteins are known to accumulate highly during seed development or in tissues subjected to water-deficit. LEA proteins are thought to protect proteins and biomembranes under water-deficit conditions. We have identified a novel gene (WCI16) that is induced during cold acclimation in winter wheat. WCI16 did not show any sequence similarity to known classes of LEA proteins. Here, we show that WCI16 is a novel LEA-class protein that is involved in freezing tolerance. Recombinant WCI16 protein exhibited stability after boiling and in vitro cryoprotection of the freeze-labile enzyme, L-lactate dehydrogenase. ¹H-NMR spectroscopy demonstrated that WCI16 has no hydrophobic region and forms random structures. These data suggested that WCI16 is a novel LEA-class protein. Recombinant WCI16 showed a double-stranded DNA binding activity, suggesting that WCI16 may protect DNA during environmental stresses. Overexpression of WCI16 in Arabidopsis conferred enhanced freezing tolerance over wild-type plants. These results indicated that WCI16 is involved in freezing tolerance in wheat.

View full abstract

To establish a cold culture method for a long-term preservation of patent-related microalgae, we have investigated cold response of Chlamydomonas reinhardtii as a model system. When the cells were exposed to 4^oC, high viability, more than 98%, was maintained for 7 days and then gradually decreased to 1.2% after 70 days. The surviving cells had a proliferative capacity as high as the control cells. We next performed microarray and RT-PCR analyses to investigate cold-responsive genes. The results suggested that a diverse array of genes, including genes for photosynthetic proteins, metabolic enzymes, and transcription factors, were up- or down-regulated by cold treatment. We also found the up-regulation of genes involved in circadian rhythm. Interestingly, an inseparable relationship between circadian rhythm and cold response has been demonstrated in Arabidopsis. These results suggest that microalgae may possess a cold adaptation system at least in part similar to higher plants.

View full abstract

We previously isolated a combination of high-light and heat-shock stress (HL+HS)-inducible genes, including a suppressor of the G2 allele of skp1 (Sgt1a) from Arabidopsis. Arabidopsis contains two Sgt1 genes (Sgt1a and Sgt1b). Here we explored the physiological function of Arabidopsis Sgt1a or Sgt1b under stressful conditions. The transcript and protein levels of Sgt1a markedly increased under not only HL+HS stress, but also HS stress or H₂O₂ treatment. There was no significant difference in acquired thermotolerance between the wild-type, Sgt1a-overexpressing or Sgt1b-overexpressing plants or Sgt1b-disrupted mutants (edm1). However, the acquired thermotolerance was lower in the knock-out Sgt1a mutants (KO-Sgt1a) than the wild-type plants. Furthermore, the transcript levels of some heat shock proteins (Hsp) were significantly lower in the KO-Sgt1a mutants than the wild-type plants and edm1 mutants. The present findings indicated Sgt1a to be important for the acquired thermotolerance.

View full abstract

When plants are exposed to various abiotic stresses, they respond and adapt to these stresses. DREB1A/CBF3 is one of cold-inducible genes encoding transcription factors in Arabidopsis. Overexpression of DREB1A caused increased tolerance to cold stress in Arabidopsis, suggesting that the DREB1A protein functions in cold stress-responsive gene expression. Many protein products regulated by DREB1A are probably responsible for the stress tolerance of plants.
In this study, we analyzed genes for novel membrane protein family, COR413s whose expression are regulated by DREB1A. We confirmed that these COR413 genes, COR413-IM1, COR413-IM2.1, and COR413-PM1 are cold inducible genes by using RNA gel blot analysis. We observed the subcellular localization patterns of these proteins by using GFP fusion constructs. COR413-IM1 and COR413-IM2.1 were localized at the chloroplast membrane, while COR413-PM1 was thought to be localized at ER. We generated transgenic Arabidopsis plants overexpressing these proteins. We report the phenotypic characteristics of these transgenic plants and T-DNA insertion mutants of these genes.

View full abstract

DREB2s are transcription factors that interact with a cis-acting DRE sequence and activate the expression of downstream genes involved in water- and heat shock-stress responses and tolerance in Arabidopsis thaliana. Previously, we characterized five DREB2-type genes in rice. We reported the expression patterns of these genes under abiotic stress conditions, and the subcellular localization and transcriptional activation activity of their translational products in protoplasts. Only OsDREB2A and OsDREB2B showed abiotic stress-inducible gene expression, and the functional form of the OsDREB2B mRNA accumulated significantly during stress conditions. In addition, OsDREB2B showed clear nuclear localization and the highest transactivation activity. In this study, we analyzed transgenic A. thaliana plants overexpressing OsDREB2B. They showed enhanced expression of DREB2A target genes and improved drought- and heat shock-stress tolerance. These results suggest that OsDREB2B is a key gene that encodes a stress-inducible DREB2-type transcription factor that functions in stress-responsive gene expression in rice.

View full abstract

An Arabidopsis transcription factor gene, DREB2A, is known to play an important role in environmental stress responses. DREB2B was isolated as a DREB2A homolog with the highest similarity. However, the function of DREB2B under environmental stress conditions is still unclear.
Expression analysis using qRT-PCR revealed increased expression levels of DREB2B under drought, heat and high salinity stress conditions. Transactivation analysis using a mutated DREB2B lacking the sequence which is very similar to the DREB2A negative regulatory domain (NRD) revealed that the mutation could increase the transcriptional activity of DREB2B. However, the increase in the transcriptional activity was much smaller than that observed when a corresponding mutation was induced to DREB2A. Confocal microscopic analysis of transgenic plants overexpressing GFP-fused wild-type or mutated DREB2B indicated that both proteins were localized stably in the nucleus. A consistent result was also obtained by immunoblot analysis. These data suggest that the DREB2B protein is more stable than the DREB2A protein. We are currently analyzing phenotypes of DREB2B overexpressors such as morphology and stress tolerance.

View full abstract

DREB2A is an important transcription factor that is involved in heat or water stress-inducible gene expression of Arabidopsis thaliana. It has been shown that DREB2A is selectively degraded under non-stressful conditions, whereas it becomes stable under stressful conditions. Deleting the negative regulatory domain from DREB2A turns the protein into a constitutively active form (DREB2A CA), which is more stable than the wild type protein. However, mechanisms stabilizing DREB2A in response to environmental stresses have been uncovered.
We applied heat stress to A. thaliana mesophyll protoplasts that were transiently expressing DREB2A or DREB2A CA under the control of a constitutive promoter. Immunoblot analysis showed that the accumulation level of DREB2A was significantly increased in response to heat stress. Unexpectedly, we found that even the accumulation level of DREB2A CA was increased in response to heat stress. This result suggests that DREB2A CA, as well as DREB2A, might be degraded under non-stressful conditions in leaves. Taking advantage of the protoplast system, we are now testing effects of various stress treatments or inhibitors on the stability of the DREB2A proteins.

View full abstract

Excessive accumulation of Na⁺ in plants disturbs cellular functions and inhibits K⁺ uptake under salt stress condition. It is supposed that Na⁺ and K⁺ homeostasis is crucial for plant salt tolerance. HKT-type transporters mediate uptake of K⁺ and Na⁺, and play important roles in salt tolerance by removing Na⁺ from xylem sap and maintaining K⁺/Na⁺ homeostasis. Previous functional characterization of rice HKT transporter in yeast expression system have shown that OsHKT2;2 which was isolated from salt-tolerant cultivar (cv Pokkali) functions as a Na⁺/K⁺ co-transporter and a single amino acid mutant of Na⁺ transporter OsHKT2;1 (OsHKT2;1S88G) mediates high affinity-K⁺ transport in yeast.
In this study, we carried out a functional analysis of these HKT transporters using transgenic rice plants. When overexpressed in rice, OsHKT2;1S88G increased K⁺ content in leaves under low K⁺ and high Na⁺ condition. Also overexpression of OsHKT2;1S88G and OsHKT2;2 resulted in higher K⁺/Na⁺ ratio compared with control. Our results suggest that these transporters mediate K⁺ uptake and contribute to K⁺/Na⁺ homeostasis under salt stress condition.

View full abstract

To identify rice genes that confer tolerance to environmental stresses, we are taking advantage of FOX Hunting System. In terms of physiology, abiotic stresses such as drought, salinity, heat or cold stress, provoke overlapping responses in plants, indicating the mechanisms of tolerance to these environmental stresses may share the same machinery. In this study, we are planning to perform two types of screening. The first one is "comprehensive FOX hunting" (c-FOX), where transgenic rice lines containing rice full-length cDNA are screened at T0 and/or T1 generations under salinity stress condition. We attempt to screen 2232 T1 transgenic lines, kindly provided by NIAS, and 215 independent T0 lines. The second strategy is "targeted FOX hunting" (t-FOX), for which 100 candidate genes (TOP100) are pre-selected based on the previous studies using multiple plant species, and used for examination resources to test whether overexpression of those genes confers stress tolerance under various environmental conditions. We are also evaluating existing rice cultivars with higher stress tolerance, to test as a host plant for further combinational analysis of the selected FOX genes.

View full abstract

To develop gene resources related to UVB tolerance of rice, UVB resistant rice cultivar Sasanishiki was irradiated with carbon ion beam (320 MeV: ¹²C⁶⁺, 80 Gy). In this study, we focused two isolated lines, UVB resistant mutant UVTSa-319 and UVB sensitive mutant UVSSa-1. These mutants have similar levels of the amount of UV-absorbing compounds and the repair activities of cyclobutane pyrimidine dimers and (6-4) photoproducts which have been thought as factors related to UVB tolerance to Sasanishiki. We performed Array CGH to estimate mutated region. As a result, we found that approximate 40 kbp on chromosome 7 containing 2 genes (AK111251, AK071492) which have unknown function was deleted in UVTSa-319. In UVSSa-1 mutant, the region on chromosome 7 containing 2 genes (AK073834, AK121807) was deleted. Next, to deduce the relationships between deleted genes and UVB tolerant mechanism in these mutants, microarray analyses were performed using RNAs of Sasanishiki and these mutants. In this meeting, we discuss about these functions of deleted genes and relationships of UVB tolerance or sensitivity.

View full abstract

Thioredoxin (Trx) is a ubiquitous protein, which supplies the reducing equivalents to the target proteins to maintain the redox conditions in the cell. In general, cyanobacterium has two Trx reducing pathways; NADPH-Trx reductase (NTR) pathway and Fd-Trx reductase pathway. Based on the analyses of the NTR and FTR disruptants of Synechocystis sp. PCC6803, NTR was clarified to be critical for the antioxidant system, though the terminal proteins of two Trx reductase pathways were not known yet (Hishiya et al. 2008). In this study, we compared the oxidized proteins in the wild cells with those in the Trx reductase disruptants by 2D-electrophoresis following the fluorescence labeling of the oxidized proteins. Consequently, we successfully detected several proteins observed especially in the mutant cells. In addition, difference of the protein expression level in the mutant cells under the oxidized conditions was investigated by conventional 2D-electrophoresis. Based on these studies, the difference of two Trx reductase pathways in cyanobacteria is discussed.

View full abstract

Elongation factor G (EF-G), a key protein in translational elongation, is sensitive to oxidative stress. We have recently found that EF-G is inactivated upon oxidation of two specific Cys residues and subsequent formation of an intramolecular disulfide bond in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we examined the role of EF-G (Slr1463) in the synthesis of proteins under strong light that causes oxidative stress. In a mutant that overexpresses EF-G, the synthesis of the D1 protein de novo is enhanced under strong light, whereas the synthesis of most of other proteins de novo was repressed. In this mutant, the level of another EF-G homolog Sll1098 was decreased. These observations suggest that EF-G (Slr1463) might be specific to the light-dependent synthesis of proteins.

View full abstract

To investigate the involvement of reactive oxygen species (ROS) derived from chloroplasts in plant response to environmental stress, we have created the system for transient suppression of thylakoid membrane-bound ascorbate peroxidase in Arabidopsis plants. Microarray analysis using this system revealed that ROS derived from chloroplasts affect the expression of approximately 800 genes, including the genes involved in response to plant hormones and abiotic and bitic stresses. To clarify the molecular mechanism of oxidative signaling derived from chloroplasts, we isolated and characterized paraquat-induced photooxidative stress sensitive and insensitive mutants (pss and psi, respectively) from knockout lines, which are disrupted genes responsive to ROS derived from chloroplasts. Among 109 lines, three psi (psi1~ 3) and one pss (pss1) mutants were isolated. The transcript levels of PSS1 and PSIs were suppressed at early-stage after treatment with paraquat, but were induced at late-stage.

View full abstract

The tropospheric ozone (O₃), which is the major component of photochemical oxidants, is considered as the most phytotoxic air pollutant. High concentration of O₃ causes significant damage in plant growth and induces oxidative stress that activates programmed cell death. Analysis of transcriptome has provided important information on understanding ozone stress response and mechanism. However, mechanisms of the ozone stress response in plants remain to be clarified. In this study, we screened our CRES-T transgenic plants, in which the chimeric repressor against various transcription factor is expressed, to identify transcription factors involved in ozone stress response. CRES-T is a gene silencing technology, which could efficiently suppress functions of both target and functionally-redundant transcription factors. We describe the trascription factors that are responsible for O₃-sensitive or insensitive phenotypes of CRES-T transgenic plants.

View full abstract

One of the most important responses of plants to hypoxic stress is aerenchyma formation in roots. However, it is difficult to visualize aerenchyma by live imaging using fluorescence technique because aerenchyma itself is a large intercellular space. Synchrotron X-ray computed tomography (CT) is previously shown to be useful for nondestructive observation of intercellular spaces in pome fruits. In the present study, we have tried nondestructive observation of aerenchyma in rice (Oryza sativa L. ssp japonica) primary roots using X-ray CT at BL20B2 beam line of SPring-8. Rice seeds were sandwiched between two agar plates, only one of which contained 270 mM mannitol, and roots were allowed to grow for four days attaching to both agar plates. Aerenchyma was clearly shown to develop in a side of a root treated with mannitol using X-ray CT and 3D modeling, confirming that this technique is useful for the nondestructive observation of aerenchyma. Next, primary roots grown in agar medium were examined using X-ray CT. Using this technique, almost entire three-dimensional connection of aerenchyma from the basal to the distal part of the roots was shown in situ.

View full abstract

Plants accumulate anthocyanins in response to drought stress. We found that accumulation of anthocyanins was reduced in ABA-deficient and -insensive mutants, and was enhaned in ABA-overaccumulation mutants under drought stress conditions. Expression of genes for flavonoid biosynthesis or its regulators was induced by drought stress treatment. Drought-induction of these genes was observed even in ABA-deficient mutants and was synergistically enhanced in ABA-overaccumulation mutants. These results suggest that drought-induced anthocyanin accumulation is regulated by both ABA-dependent and –independent mechanisms. We examined whether anthocyanins function in detoxification of reactive oxygen species (ROS) accumulating in response to drought stress. DAB staining was intense in ABA-deficient and was weak in ABA-overaccumulation mutants after drought stress. In addition, transgenic plants that overaccumulate anthocyanins showed drought stress tolerant. These results suggested that anthocyanins function as an antioxidant against drought-induced ROS. Now, we are analyzing the drought stress tolerance and the levels of ROS of several anthocyanin-deficient mutants.

View full abstract

Plant cells respond to the environmental stress through reversible posttranslational modifications of important signal molecules. The dynamic glycosylation of serine or threonine by O-linked β-N-acetylglucosamine (O-GlcNAc) is critical to many cellular response and reciprocal to O-phosphorylation. In Arabidopsis, SPINDLY (SPY) gene encodes an O-GlcNAc transferase (OGT), which was originally identified as a negative regulator in GA signaling. In this study, we found that spy mutants are more tolerant to osmotic stress, such as high salinity and dehydration. SPY gene expression is specifically drought inducible. Transcriptome analysis of spy-3 mutant discovered that the expression of some drought-inducible genes, in addition to many GA inducible genes, was upregulated. More evidently, in 2h dehydrated spy-3 plants, the expression of many drought-responsive genes, especially LEA protein genes, is higher than that in wild-type plants. In addition, some DREB2A specific downstream gene expressions were enhanced in spy-3 mutant, in early dehydration stress.

View full abstract

Using DNA microarray from a terrestrial cyanobacterium Anabaena sp. PCC7120, the typical desiccation-responsible genes were selected and the gene-disruptant were characterized. All of the disruptants showed low viability under desiccation using cells grown in nitrogen-free medium. These results may suggest that desiccation-tolerant genes contain function in N₂-fixation, are expressed irrespective of nitrogen content to protect desiccation sensitive N₂-fixing heterocyst and express to stabilize intra- and outer-cellular condition under desiccation.
Desiccation tolerant of N₂-fixing cyanobacterium, Nostoc commune is closely relative of the Anabaena. The Nostoc has ability to use scientific research for desiccation tolerance system, food and soil for plantation. These abilities expect to improve devastating soil to nutrient-rich soil including space agriculture. So it was tried to isolate the Nostoc and succeeded to cultivate the Nostoc axenically. To confirm ability of the Nostoc soil, the Nostoc was used plantation as nutrient containing plate. The result of difference plant growth between nitrogen-deficient plate and cyanobacterial mat is now in progress.

View full abstract

Starch is an important form of carbon reserve in plants. While initiation of glycogen synthesis in yeast and animal has been well characterized, mechanism of initiating starch synthesis in plants remained largely inconclusive. The expression trend of rice (Oryza sativa L.) glycogenin glucosyltransferase (OsGGT) was tightly linked to that of starch biosynthesis during the day and night in the rice cultivar Nipponbare. While repressing OsGGT expression led to clear reduction in starch contents in GGT mutant line (ggt), transgenic rice plants overexpressing OsGGT showed 1.3 folds higher starch contents compared to WT plants. Moreover, after two days of complete submergence, OsGGT overexpressing plants maintained 61% of its original starch contents prior to submergence compared to only 34% in WT plants. These results provide supporting evidence that OsGGT is the starch initiating protein in rice and its overexpression provide new strategy to increase starch contents in rice with greater tolerance to submergence

View full abstract

The dune grass Leymus mollis grown in the seaside is a wild relative of wheat (Triticum aestivum) and is important genetic resource for improving wheat stress tolerance. We used Subtractive Subtraction Hybridization to identify drought and salinity responsive genes from L. mollis. Twenty one genes were identified and confirmed to be differentially regulated. Chloroplast inositol phosphatase (CIP), phosphoethanolamine methyltransferase (PEAMT), ETTIN-like auxin response factor (ARF) and unknown gene were among the highly regulated under drought stress; while allene oxide cyclase (AOC) and methyljasmonate induced lipoxygenase (LOX) genes were among the highly responsive genes under salt stress. These genes were also shown to be differentially regulated in response to jasmonic or abscisic acid. Southern analysis indicated that L. mollis genome has higher number of the isogenes for PEAMT, CIP and ARF compared to that found in the Chinese spring wheat. Identification of these genes represents important genetic resources for wheat improvement

View full abstract

Despite its indispensable function in the symbiotic association between legumes and rhizobia, for the vast majority of plants, the physiological role of purine metabolism remains elusive. We have previously demonstrated that RNA interference-mediated suppression of xanthine dehydrogenase (XDH), the rate-limiting enzyme in purine degradation, causes defects in normal growth and development of Arabidopsis thaliana. Here, we investigated a possible role of XDH in drought tolerance, because this enzyme is also implicated in stress responses and acclimatization of plants. Compared with wild-type seedlings, XDH-suppressed lines showed significantly reduced growth and chlorophyll content after drought shock, which accompanied increased cell death and higher H₂O₂ levels. Such drought-hypersensitive phenotype of XDH-suppressed lines, however, was restored by exogenous supplementation of urate, the metabolite of XDH catalysis. These results suggest the importance of XDH in the acclimatization to drought, possibly through production of purine metabolites, such as urate and ureides, with antioxidant capacity.

View full abstract

Roots display tropisms in response to environmental cues such as gravity, light, physical contact, and moisture gradients. Hydrotropism is a directed growth towards higher water potential or moisture, and has long been thought to play an important role in drought avoidance. But, it was difficult to separate hydrotropism from gravitropism because of the constant presence of gravity. We recently developed systems to study hydrotropism of Arabidopsis thaliana, and isolated an ahydrotropic mutant, miz-kussei1 (miz1), and identified MIZ1 as a gene responsible for hydrotropism. MIZ1 encodes a unique domain (MIZ domain), which is highly conserved among terrestrial plants, but not in known genomes of other organisms such as green and red algae, cyanobacteria, or animals. Here, we found three MIZ1 homologues, PpMIL(MIZ1-Like) 1-3 in the moss Physcomitrella patens. With the gene-targeting method, we established the disruptant line for each PpMIL gene, the PpMIL1/2 double and the PpMIL1/2/3 triple disruptant lines. We are now analyzing the phenotypes of these disruptant lines.

View full abstract

Rush are monocotyledon tolerant to high salt conditions, found growing along marsh and seashore. There are few reports on the mechanism of salt tolerance in the rush. To analyze the molecular mechanisms for salt tolerance in rush, functional screening of cDNAs encoding proteins which may important role for salt tolerance in the halophytes was done using Escherichia coli as a host organism. In this screening, cDNAs encoding proline-rich arabinogalactan protein homolog were successfully isolated. Proline-rich protein may plays an important role for salt tolerance mechanism in rush.

View full abstract

Cadmium (Cd) is one of toxic heavy metals. To reduce Cd accumulation in crop plants, it is necessary to elucidate mechanisms of Cd transport and accumulation in plants. However, these mechanisms are not fully understood so far. Glutathione (GSH), a low weight tripeptide, is involved in many aspects of metabolism. In our previous work, GSH concentration in the phloem sap increased by Cd treatment. These results suggested that GSH might be playing important roles in controlling Cd transport and accumulation. In this work, we investigated effects of GSH which was administered to specific organs of plants.
Oilseed rape plants were grown hydroponically in a green house for four weeks. After treatment, plants were harvested and Cd contents were measured using an ICP spectrometer. We also employed PETIS (Position Emitting Tracer Imaging System) to visualize Cd distribution in the plants. Time-series images of ¹⁰⁷Cd distribution were obtained. Cd content in shoots was drastically reduced when their roots were treated with GSH. However, Cd content in roots did not change. Reduction of Cd transport and accumulation by GSH, administered to roots, was also confirmed by PETIS.

View full abstract

Scopelophila cataractae (Mitt.) Broth. is one of the interesting moss that often grows under copper rich environments, such as under copper roofs of temple, and this moss accumulates copper in the body. However, physiological knowledge about a mechanism of copper tolerance and style that expands distribution of this moss is still almost unknown. Thus, we are analyzing the response to the copper of S.cataractae. First of all, to elucidate the response to the copper, we examined the effect of the copper treatment on the protonema growth. As a result, compared with control, the diameter of the 14 day cultured protonemal colony has increased by the copper treatment (up to 800μM). In contrast, number of protonamal gemma formations has increased in the control. These result suggest that S.cataractae has the ability to change the morphogenesis according to the environmental copper concentration.

View full abstract

In Arabidopsis thaliana , vacuolar membrane Metal Tolerance Protein 1 (MTP1) plays important role for zinc homeostasis. We reported that AtMTP1 is a Zn²⁺/H⁺ exchanger and involved in zinc tolerance in plants by transporting excess cytoplasmic zinc into vacuoles. Furthermore, we demonstrated that deletion of a His-rich loop caused marked increase in V_max value and proposed that the His-rich loop is essential for maintenance of t the cytoplasmic zinc level (JBC, 283: 8374-8383, 2008; PCP, 50: 1156-1170, 2009). In this study, we made Arabidopsis plants overexpressing AtMTP1 or AtMTP1Δ185-216 and analyzed metal tolerance under zinc excess conditions and zinc distribution in cells by using a zinc fluorescent probe Zinpyr-1 in order to elucidate physiological role of the His-rich loop. We also examined differences in protein stability, ion selectivity and zinc transport ability between AtMTP1 and AtMTP1Δ185-216 in A. thaliana. We will discuss about molecular function and physiological role of the His-rich loop.

View full abstract

Acacia mangium, a leguminous tree, shows tolerance to aluminum (Al) and can vigorously grow in acid soils in wild conditions. Previously (at the 50th annual meeting), we detected low-pH responsible genes using differential display reverse transcription PCR (DDRT-PCR) analysis and semi-quantitative PT-PCR analysis in cell suspensions of A. mangium that were induced from hypocotyls. Full-length cDNA cloning of those low-pH responsible genes are now in progress.
In addition to proton (H⁺) toxicity, Al stress is the most significant factor that limits plant growth in acid soils. So the effects of Al on cell growth of A. mangium and gene expression patterns of the low-pH responsible genes under Al stress were examined. Our results indicated that several genes showed differential expression patterns between low-pH stress and Al stress.
Furthermore, degenerate PCR approaches using primers designed from known Al tolerance rerated genes such as aluminum activated malate transporter (ALMT) and aluminum activated citrate transporter (AACT) allowed us to detect the homologous genes of A. mangium. We also report the results of expression analysis of these genes.

View full abstract

The gene of Sll1558 in Synechocystis PCC 6803 was identified as up-regulated gene by microarray analysis under the acid stress condition (pH 3.0). Relative expression levels of the gene at 0.5, 1 and 4 hours were 3.51, 6.94 and 4.05 respectively. The gene encodes mannose-1-phosphate guanylyltransferase (EC 2.7.7.13) which catalyses D-Mannose 1-phosphate to GDP-D-mannose and involves N-glycan biosynthesis.
In the Synechocystis genome, sll1496 was also annotated as the enzyme. In this study, deletion mutants of the genes were constructed. The deletion mutants of sll1558 were more sensitive to acid stress compared with wild type cells. These mutants also display increased sensitivity to salt (0.5M NaCl) and osmotic (0.5M sorbitol) stress. Real-time PCR analysis of the levels of the transcripts in the wild type cells and mutants will also be reported.

View full abstract

In previous presentations we have shown the effects of metal hydride water on plants. Cut flowers that absorb this water display unusual phenomena, which include a tendency to grow higher and continue to produce new flowers or leaves. Plants commonly maintain their condition for a long period of time while others mysteriously wither and die suddenly.
Flowers will usually begin to droop a short time after being cut. However, flowers that have absorbed the pH and ORP altered metal hydride water tend to maintain their vitality. This phenomenon appears to be caused by an ionic hydride reaction between CaH₂and hydrogen in the form of a minus hydrogen ion. Changing the concentration of available minus hydrogen ions seems to control growth acceleration, plant preservation and flowering. This holds the possibility of controlling hydroponic plant health and growth, not only for cut flowers but also for plants in the agricultural field.
We are now clarifying under what conditions these phenomena occurs. We will report on research still in progress and discuss why similar phenomena are unlikely to occur in other forms of alkaline ionized water at a similar pH.

View full abstract

Jasmonates (JAs) are essential signal molecules modulating the plant response to biotic and abiotic stresses as well as several growth and developmental traits. COI1 has a crucial role in the JA-signaling pathway. COI1 encodes an F-box protein that associates with Skp/cullin/F-box (SCF) E3-ubiquitin ligase. E3-ubiquitin ligase are involved in the ubiquitination of target proteins for degradation by 26S proteasome. JAs have been suggested to promote ubiquitination and degradation of negative regulators of JA signaling. Recently, it reported that COI1 protein directly binds to JA-Ile and serves as a receptor. In this study, we attempted to analyze JA-signaling pathway in rice.

View full abstract

Plants are exposed to various stresses, such as pathogen attacks and environmental stress. To adapt to the stressful environment, plants have evolved unique self-protection systems. In plants, salicyclic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) play important roles in adaptation to biotic and abiotic stresses. In addition, recent studies have shown that each plant hormone is mutually antagonistic interactions in complex networks of various signaling pathways. For instance, it has revealed that treatment with ABA suppresses the induction of systemic acquired resistance (SAR) by inhibiting the pathway both upstream and downstream of SA, independently of the JA/ethylene-mediated signaling pathway. Conversely, the activation of SAR suppresses the expression of ABA biosynthesis-related and ABA-responsive genes. These results show that antagonistic crosstalk occurs at multiple steps between the SA-mediated signaling of SAR induction and the ABA-mediated signaling of environmental stress responses. Here, we will discuss that involvement of plant hormones and plant hormone mediated-gene expression in SA-, JA- and ABA-treated leaves.

View full abstract

Root-knot nematodes (RKNs) are a major parasite of plants, causing approximately 5% loss of total agriculture world-wide. RKN are sedentary endoparasites that establish a single permanent feeding site within plant roots as a juvenile, and then spend their whole remaining life-cycle at this single site. This is achieved by inducing normal plant cells to change into a new specialised cell-type, a Giant Cell (GC), that functions as a nutrient transfer cell. The pathway for formation of a GC from a normal plant cell remains unclear, mainly due to the difficulties in directly studying the early infection stages hidden within the host root. Using the tomato variety Micro-Tom as a model system, we have initiated a forward genetics approach to identify host genes involved in RKN-induced GC formation and are also studying the early GC cells directly. Lab home page: http://gotolab.cris.hokudai.ac.jp

View full abstract

Leguminous plants form nodules and establish symbiosis with rhizobia. Inverted Repeat Lacking Clade (IRLC) legumes form indeterminate nodules. Rhizobia in nodules (bacteroids) of IRLC legumes show morphological and physiological changes compared to free-living bacteria. It is suggested that the differentiation of bacteroids are induced by Nodule specific Cysteine Rich peptides (NCRs) derived from host plants. Lotus japonicus, Glycine max, and Phaseolus vulgaris, which do not belong to IRLC, form determinate nodules, in which characteristics of bacteroids are as same as cultured bacteria, and NCRs do not exist. Leucaena glauca is a species branched early taxonomically in legumes and forms indeterminate nodule. However, characteristics of bacteroids in L. glauca are not investigated. In this study, microscopic observation and detection of NCRs in L. glauca bacteroids were performed to discuss about relations between nodule type, bacteroid characteristics and NCRs. Drastic changes of bacteroids like as IRLC legumes were not observed, and NCR peptides were not detected in L. glauca. These results suggest that the bacteroidal differentiation by NCR peptides is specific to IRLC legumes.

View full abstract

Type III secretion systems (T3SSs) play an essential role in the pathogenicity of many bacterial pathogens. Phytopathogenic bacteria use the T3SS to deliver effector proteins directly into host plant cells to promote pathogenicity. To counteract, plants have evolved resistant proteins to monitor the effectors and halt pathogen invasion and disease. We have investigated the T3SS of Mesorhizobium loti, a microsymbiont of model legume Lotus japonicus. Expression analysis showed that T3SS is expressed in response to host-derived flavonoids, suggesting that it plays a role during infection. Depending on host species, T3SS affected symbiosis either positively or negatively. By inoculating mutants of various effector candidates, an ORF mlr6361 was identified as a major determinant of nodulation restriction. Mlr6361 is a protein of 3,056 amino acids containing 15 repetitions of a sequence motif of 40 to 45 residues and a shikimate kinase-like domain at its carboxyl-terminus. Homologues of Mlr6361 are present in several plant pathogens. These results suggested that some legume recognize Mlr6361 as potentially pathogen-derived and subsequently halts the infection of symbiotic partner.

View full abstract

Lipopolysaccharide (LPS) of Gram negative bacteria is recognized by plants as one of the PAMPs (Pathogen Associated Molecular Patterns) and is also involved in establishing normal symbiosis between rhizobia and their host legumes. However, the recognition mechanism is still not clear in plants. LPS binding protein (LBP) forms a complex with LPS and activates innate immune system in animals. On the genome of Lotus japonicus, four genes, LjLBP1, 2, 3 and 4, were identified as candidates for plant LBP and their expression were analyzed with or without microsymbiont Mesorhizobium loti. Among the LBPs, LjLBP3/4 was up-ragulated transiently at 4h after inoculation of M. loti. However, the expression ofLjLBP3/4 was strongly repressed in nodulated plants compared with that of plant supplied with 0.5 mM NH₄Cl. These results indicate that the expression of each LBP is regulated in different manner depending on the stage of the symbiosis. Considering with the symbiotic phenotype of LBP mutant lines of L. japonicus, LBPs will be involved in legume-Rhizobium symbiosis.

View full abstract

Legume plants establish nitrogen-fixing nodules with rhizobia in a species-specific manner. During the nodulation process and symbiotic nitrogen fixation (SNF), a large variety of metabolites are exchanged between plant cells and rhizobia via membrane transport. ATP binding-cassette (ABC) proteins, which constitute the largest transporter family in plants, may play important roles in these processes, and indeed several members of ABC protein gene are strongly expressed with different induction patterns during the nodulation formation.
Tissue specific expression study was done with real time PCR analysis to demonstrate that several members of the full-length ABCG subfamily are strongly up-regulated by rhizobial infection. cDNA array analysis during nodulation (Kouchi et al., DNA Research, 11: 263-274, 2004) suggested that one gene chr3.CM0026.74, which was designated as LjABCG1, was most prominently up-regulated in the early stage of infection.

View full abstract

Soybean (Glycine max) is one of the world's leading protein and oil crops. The mechanism of nutrient uptake from the soil is an important matter. Approximately two-thirds of all species of land plant are symbiotically associated with arbuscular mycorrhizal (AM) fungi in the roots. Fungal hyphae can explore the soil, efficiently take up nutrients from the soil and transfer to the roots of host plants. In colonized root cortical cells, fungal intraradical hyphae spread in the intracellular spaces and temporally form characteristic symbiotic structures, arbuscules, where fungal hyphae are highly branched to extend their surface area. Although it is assumed that arbuscule is the site of major Pi transfer to the plants, there is no direct evidence that ammonium is also transferred at this structure. Therefore, to know the mechanism of ammonium uptake in soybean associated with the AM symbiosis, we searched AMT genes in JGI Glycine max genome database with BLAST analysis, and found sixteen genes (GmAMTs). The gene specific RT-PCR indicated that three genes (GmAMT4.1, GmAMT4.3 and GmAMT4.4) were the AM-inducible candidates. We will discuss the function of them.

View full abstract