Most plants sense their environmental changes such as photoperiod by using an endogenous circadian clock to regulate their developmental phases. The core oscillator of the clock has been reported to be composed of a negative feed back loop of transcription. Meanwhile, recent biochemical studies have been shedding light on the importance of post-translational regulations for the circadian mechanisms. ZEITLUPE (ZTL) family members are unique proteins which have three characteristic domains; a LIGHT, OXYGEN AND VOLTAGE domain, an F-box domain and six kelch repeats. Recently, two of the three family members, ZTL and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 were reported to be novel types of blue light receptors which post-translationaly regulate the circadian clock and photoperiodic flowering, respectively. In this review, studies about ZTL family members making substantial progress are summarized and discussed.
Several plant basic leucine zipper (bZIP) proteins have been shown to play a role in chalcone synthase (CHS) gene expression, and some are regulated by phosphorylation/dephosphorylation. We isolated SBZ1 (Soybean bZIP protein 1) and showed that the recombinant protein binds in vitro to the 5′ region of soybean CHS1, at a sequence that confers the elicitor-inducible expression of CHS genes. The deduced amino acid sequence of SBZ1 has features characteristic of bZIP transcription factors, including a highly basic putative DNA-binding domain containing a nuclear localization sequence, as well as four domains designated D1–D4 that are highly conserved among the subfamily of bZIP factors, which includes tobacco BZI-1 and parsley CPRF2. The presence of these regions indicates that SBZ1 is a CPRF2-related bZIP transcription factor. The protein kinase inhibitor K252a blocks CHS induction in elicited soybean cells, suggesting that protein phosphorylation is involved in induction of the CHS signal pathway. Phosphorylation assays indicated that SBZ1 is phosphorylated in vitro in a soybean cell extract, and that this phosphorylation depends on Ca2+. Furthermore, recombinant soybean CDPK and the α subunit of CKII phosphorylate SBZ1 in vitro. However, unlike other related bZIP proteins, phosphorylation had no effect on either the DNA-binding activity of SBZ1. Therefore, we conclude that SBZ1 is regulated by phosphorylation, but in a different manner than are related bZIP factors.
The DnaK/Hsp70 family is a molecular chaperone that binds non-native states of other proteins, and concerns to various physiological processes in the bacterial, plant and animal cells. Transgenic tobacco plants expressing a molecular chaperone DnaK from a halotolerant cyanobacterium Aphanothece halophytica show enhanced seed yields as well as enhanced tolerances for salt and heat stresses. High-temperature treatment during the reproductive stage decreased total dry weight of seeds in both transgenic and wild-type tobacco, but more severely in the wild-type. Transgenic tobacco plants exhibited higher activities of ascorbate peroxidase and catalase than wild-type plants. Similar results were obtained for salt stress during the reproductive stage. Transgenic rice plants expressing ApDnaK was also constructed. Transgenic rice plants exhibited the enhanced activities for Calvin-cycle enzymes, and showed faster growth and higher seed yield compared with the wild-type rice under normal growth conditions. Transgenic rice plants also showed enhanced tolerance for high temperature and salt stress compared with the wild-type rice. These results suggest a relation of increased folding activity with enhanced stress tolerance, increased seed yield, and total plant biomass.
Effects of a wide range of concentrations of four salts (NaCl, KCl, MgCl2, CaCl2) on cell proliferation were investigated in suspension culture of a mangrove plant, Sonneratia alba. The results were compared to those obtained with suspension culture of another mangrove species, Bruguiera sexangula and non-mangrove tobacco BY-2 cells. Effects of salts on suspension cells were determined with both large scale (LS) method using a 100 ml culture flask, and/or a small scale (SS) method using a 24-well culture plate. Settled cell volume (SCV), packed cell volume (PCV), wet weight (WW) and dry weight (DW) were measured in LS method and PCV was in SS method. High concentrations of NaCl (25–100 mM) and MgCl2 (25 mM) showed strong stimulatory effects on growth in S. alba suspension culture, and low concentrations of KCl, MgCl2 and CaCl2 (10 mM) also showed similar stimulatory effects. In contrast, a remarkable stimulatory effect was not observed in B. sexangula and tobacco BY-2 cells. Cationic ions are effective for the growth of suspension cells in both mangrove species S. alba and B. sexangula. The SS method using multi-well culture plate and micro-tube measurement method developed in this report, can be applied for analysis of factors for stress tolerance.
Angelica acutiloba and related species have been known for their analgesic and sedative effects, and are widely used as the key ingredient in Japanese traditional Kampo medicine. Among various varieties, Angelica acutiloba var. acutiloba has long been grown in the Obuka area of Nara prefecture, Japan, and has been evaluated as an excellent variety. Recent cultivation of this and other varieties of Angelica in various regions of East Asia prompted us to identify DNA polymorphisms that distinguish the acutiloba variety from others. Random Amplified Polymorphic DNA analysis and sequencing selected genomic regions among Angelica plants of different origins identified several nucleotide changes among the varieties tested, which enabled us to distinguish the acutiloba variety from others. We also report an improved protocol for genomic DNA extraction from dried root samples of Angelica.
The circadian clock regulates a wide variety of processes including the control of photoperiodic flowering and organ elongation in higher plants. Arabidopsis is a facultative long-day (LD) plant and flowers much earlier under LD and continuous light (LL) than short-day (SD) conditions. Although many of the genes required for the control of photoperiodic flowering have been identified, the precise mechanisms underlying the recognition of critical day or night lengths required for photoperiodic responses have not been fully clarified. To address this issue, we investigated circadian outputs in the loss-of-function of PSEUDO-RESPONSE REGULATOR (PRR) genes, which are believed to be clock components, under LD and LL. Here we report that prr9 flowered earlier under LL but later under LD compared to wild-type plants, which showed an opposite control of flowering response under these conditions. Although flowering times under LD and LL were similar, prr9;prr7;prr5 mutant plants showed an opposite control of petiole elongation under LD and LL. Under LL, the prr9;prr7;prr5 mutant plants had shorter petioles but longer hypocotyls than those of wild-type plants. Based on our results, we propose some models to explain the organ-specific effect caused by mutations in Arabidopsis clock genes.
For a global analysis of gene expression during nodule tissue formation in a model legume, Lotus japonicus, a cDNA array was constructed using 18,144 non-redundant clone set deduced from 3′-end expressed sequence tags (EST, Kouchi et al. 2004). In the current study, to obtain peptide coding information from 5′-ends of clone, we generated 7,320 5′ EST sequences. BLAST search against a protein database indicated that 65.5% of the ESTs showed significant similarity to registered sequences. We identified numerous of nodule-enhanced genes, including many involved in diverse cellular processes, suggesting that the EST resources generated in this study will be a useful tool for the identification of genes related to nodulation and symbiotic nitrogen fixation.
Two cytoplasmic male sterile (CMS) lines were found in the progenies of an intergeneric cybrid (Brassica oleracea L. var. capitata) derived from asymmetrical protoplast-fusion and an interspecific chimera (Brassica rapa L. var. peruviridis) derived from in vitro grafting. Although these CMS lines were different in origin, they both had abnormal anthers and aborted pollen grains. The mitochondrial DNA in these CMS lines indicated the presence of orf138, an indicator gene for Ogura-type cytoplasm. In this study, direct sequencing of two regions near the rps16 in chloroplast DNA was performed. Although the sequence of the two regions in these two CMS lines was different from that in their parental lines, it was the same as the sequence in an Ogura-type CMS radish. Thus, these results suggest that the two CMS lines possess chloroplast DNA of the Ogura-type or a similar type as well as Ogura-type mitochondrial DNA. Furthermore, the region between rps16 and trnK was compared among species related to Brassica by PCR. The result indicated the presence of many sequence variations in this region and suggested that chloroplast DNAs could be distinguished by analyzing this region by PCR.
Host-pathogen interactions were investigated on a Japanese birch (Betula platyphylla var. japonica, Tohoku) plantlet after infection with a canker-rot fungus, the Inonotus obliquus IO-U1 strain. For a time-course study, intact, wounded, and infected plantlets were collected from 2 h to 30 days after treatment. Notable changes were observed morphologically in the treated portion of wounded and infected plantlets. Phenolics first deposited at the cut margin and subsequently in vessels after 4 h of infection. Their deposition extended to other xylem elements, the cortex, and the pith with an increase in the infection period. Phenolics deposition was extensive at 10 days post-inoculation (dpi), when most of the cells were entirely filled with phenolics. A necrophylactic periderm (NP) was formed at the junction of the original periderm with a layer of 2–4 new phellem cells at 30 dpi. Based on the results obtained, phenolics deposition and NP formation are considered to occur as infection-induced responses in Tohoku birch plantlets infected with I. obliquus IO-U1.
Glutathione S-transferase (GST) plays an important role in the transport and accumulation of anthocyanin and proanthocyanidin in plants. In our previous study on Arabidopsis thaliana overexpressing the PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) gene encoding an MYB transcription factor, the AtGSTF5 and AtGSTF6 genes encoding GST-like protein were up-regulated along with TRANSPARENT TESTA 19 (TT19), which is required for the accumulation of anthocyanin and proanthocyanidin. The proteins encoded by these 3 genes showed very weak GST activities as detected by using recombinant proteins expressed in Escherichia coli. The anthocyanin levels were severely decreased in the tt19 mutant but not in the Atgstf6 mutant, suggesting that TT19 is almost exclusively involved in anthocyanin accumulation. The results of co-expression network analysis using public transcriptome data corresponded to the proposition of the predominant role of TT19 in anthocyanin accumulation.
Hairy roots induced by Agrobacterium rhizogenes-mediated transformation are widely used for the study of metabolic regulation and large-scale metabolite production. We developed an alternative root culture using an allelic mutant of Arabidopsis superroot 1 (sur1). When the mutant was cultured in liquid modified Heller's medium developed for hairy root cultures, it grew rapidly into a globe-shaped rooty phenotype and was easily subcultured in the medium. This mutant is advantageous as the desired characteristics can likely be maintained via seeds because the rooty phenotype is recessive. To verify this postulate, heterozygous sur1 plants were crossed with fatty acid desaturase (fad) mutants. The sur1 homozygous mutants under fad2 and fad3 background accumulated high amounts of oleic acid and linolenic acid, respectively, and the mutant crossed with a FAD3 overexpression transformant accumulated high amounts of α-linolenic acid. Thus, the sur1 root culture system is an alternative tool for studying metabolic regulation and the production of useful compounds in roots.
Ipomoea aquatica (water spinach) is a common aquatic plant growing in lakes and wetlands in Southeast Asia. Due to its vigorous growth, they were considered to be potentially useful for remediation of polluted water with, for example, high sulfate and heavy metals. In previous studies, we successfully constructed transgenic I. aquatica plants, which simultaneously expressed two genes encoding serine acetyltransferase and cysteine synthase involved in sulfate assimilation pathways. Resulting transgenic plants were shown to rapidly grow and to accumulate sulfate at a high level. In the present study, we tested the effect of cadmium on their physiological and biochemical features. Upon hydroponical cultivation in the presence of 200 μM cadmium for 7 days, two transgenic lines (SR1 and SR2) accumulated 2- to 4-fold higher levels of cysteine and glutathione than the wild type control plants. When plantlets were exposed to 100 μM cadmium for 30 days, wild type and transgenic SR2 plantlets died, or growth was greatly retarded with reduced biomass, whereas transgenic SR1 exhibited a 1.7-fold increase in total biomass in comparison with the initial weight at day-0 of cadmium treatment. These results suggested that some transgenic plants expressing serine acetyltransferase and cysteine synthase could mitigate detrimental effects of cadmium toxicity, perhaps by efficiently producing and accumulating sulfuric compounds.
In order to generate transgenic radish (Raphanus sativus L., cv. Jin Ju Dae Pyong), hypocotyl explants were cultured on Murashige and Skoog medium containing 4 mg l−1 AgNO3, 5 mg l−1 acetosyringone, 4 mg l−1 6-benzyladenine, and 3 mg l−1 α-naphthaleneacetic acid in addition to either 10 mg l−1 hygromycin or 100 mg l−1 paromomycin after co-cultivation with disarmed Agrobacterium tumefaciens harboring a plant expression binary vector. Explants co-cultivated with A. tumefaciens GV3101 harboring pCAMBIA1301 and A. tumefaciens EHA101 harboring pPTN290 produced putative transgenic adventitious shoots at frequencies of 0.26% and 0.18%, respectively. Northern blot analysis revealed the gus gene transcript was detected in 8 regenerated plants which confirmed their genetic transformation. The transgenic plants were grown to maturity after vernalization in a greenhouse and appeared morphologically normal. Progeny analysis of independent transgenic plants demonstrated that the gus gene was transmitted in a Mendelian pattern in 3 lines, indicating a single copied gene was incorporated into the genome.