Overexpressing plant ferredoxin-like protein enhances photosynthetic efficiency and carbohydrates accumulation in Phalaenopsis.

Crassulacean acid metabolism (CAM) is one of three major models of carbon dioxide assimilation pathway with better water-use efficiency and slower photosynthetic efficiency in photosynthesis. Previous studies indicated that the gene of sweet pepper plant ferredoxin-like protein (PFLP) shows high homology to the ferredoxin-1(Fd-1) family that belongs to photosynthetic type Fd and involves in photosystem I. It is speculated that overexpressing pflp in the transgenic plant may enhance photosynthetic efficiency through the electron transport chain (ETC). To reveal the function of PFLP in photosynthetic efficiency, pflp transgenic Phalaenopsis, a CAM plant, was generated to analyze photosynthetic markers. Transgenic plants exhibited 1.2-folds of electron transport rate than that of wild type (WT), and higher CO2 assimilation rates up to 1.6 and 1.5-folds samples at 4 pm and 10 pm respectively. Enzyme activity of phosphoenolpyruvate carboxylase (PEPC) was increased to 5.9-folds in Phase III, and NAD+-linked malic enzyme (NAD+-ME) activity increased 1.4-folds in Phase IV in transgenic plants. The photosynthesis products were analyzed between transgenic plants and WT. Soluble sugars contents such as glucose, fructose, and sucrose were found to significantly increase to 1.2, 1.8, and 1.3-folds higher in transgenic plants. The starch grains were also accumulated up to 1.4-folds in transgenic plants than that of WT. These results indicated that overexpressing pflp in transgenic plants increases carbohydrates accumulation by enhancing electron transport flow during photosynthesis. This is the first evidence for the PFLP function in CAM plants. Taken altogether, we suggest that pflp is an applicable gene for agriculture application that enhances electron transport chain efficiency during photosynthesis.

Biogeography of land snail genus Acusta (Gastropoda: Camaenidae): Diversification on East Asian islands.

East Asia has highly diverse and endemic biota due to its complex geological and climatic history and its diversified topography. The continental and insular distributions of land snail genus Acusta in East Asia provide a good opportunity to compare the evolutionary processes in this group under different biogeographical conditions. In this study, we inferred the evolutionary history of the land snail genus Acusta by a molecular phylogeny and investigated how the palaeogeographic events shaped species diversity and the distribution of the Acusta genus within the island arc. A concatenated dataset generated from sequences of one nuclear (ITS2) and two mitochondrial (16S, COI) gene fragments, include most of nominal taxa of the genus, four related species and one outgroup. We constructed the phylogeny and the evolutionary history of the genus through maximum-likelihood and Bayesian inference methods, using a Bayesian molecular clock and ancestral range estimation. Our results suggested that currently recognized species in Acusta are polyphyletic. The traditionally accepted concept of the affinity of Acusta and Bradybaena is not supported. The hypothesis of colonization via land bridges during the Pleistocene glaciations for the biota of East Asian islands is not supported. Instead, the origin and diversification of the genus Acusta was dated to the late Miocene-Pliocene from an area around North and Northeast China to South China and East Asian islands Three major evolutionary lineages were identified. Two of the major lineages demonstrate distinct evolutionary histories, as sympatric speciation is the major speciation process for the continental clade, while the insular clade originated from founder events. Taiwan functioned as an important source of diversification for species on the East Asian islands possibly through passive dispersal of different mechanisms. The sea level fluctuations caused by the Pleistocene glacial cycles play a role in the subsequent dispersion and diversification of species of the continental clade, such as the more recent range expansion of A. redfieldi from South China to Taiwan and Japan.

Overexpression of plant ferredoxin-like protein promotes salinity tolerance in rice (Oryza sativa).

High-salinity stress is one of the major limiting factors on crop productivity. Physiological strategies against high-salinity stress include generation of reactive oxygen species (ROS), induction of stress-related genes expression, accumulation of abscisic acid (ABA) and up-regulation of antiporters. ROS are metabolism by-products and involved in signal transduction pathway. Constitutive expression of plant ferrodoxin-like protein (PFLP) gene enhances pathogen-resistance activities and root-hair growth through promoting ROS generation. However, the function of PFLP in abiotic stress responses is unclear. In this study, PFLP-1 and PFLP-2-transgenic rice plants were generated to elucidate the role of PFLP under salinity stress. PFLP overexpression significantly increased salt tolerance in PFLP-transgenic rice plants compared with non-transgenic plants (Oryza sativa japonica cv. Tainung 67, designated as TNG67). In high-salinity conditions, PFLP-transgenic plants exhibited earlier ROS production, higher antioxidant enzyme activities, higher ABA accumulation, up-regulated expression of stress-related genes (OsRBOHa, Cu/Zn SOD, OsAPX, OsNCED2, OsSOS1, OsCIPK24, OsCBL4, and OsNHX2), and leaf sodium ion content was lower compared with TNG67 plant. In addition, transgenic lines maintained electron transport rates and contained lower malondialdhyde (MDA) content than TNG67 plant did under salt-stress conditions. Overall results indicated salinity tolerance was improved by PFLP overexpression in transgenic rice plant. The PFLP gene is a potential candidate for improving salinity tolerance for valuable agricultural crops.

Constitutive expression of a plant ferredoxin-like protein (pflp) enhances capacity of photosynthetic carbon assimilation in rice (Oryza sativa).

The plant ferredoxin-like protein (PFLP) gene, cloned from sweet peppers predicted as an electron carrier in photosynthesis, shows high homology to the Fd-I sequence of Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Spinacia oleracea. Most of pflp related studies focused on anti-pathogenic effects, while less understanding for the effects in photosynthesis with physiological aspects, such as photosynthesis rate, and levels of carbohydrate metabolites. This project focuses on the effects of pflp overexpression on photosynthesis by physiological evaluations of carbon assimilation with significant higher levels of carbohydrates with higher photosynthesis efficiency. In this report, two independent transgenic lines of rice plants (designated as pflp-1 and pflp-2) were generated from non-transgenic TNG67 rice plant (WT). Both transgenic pflp rice plants exhibited enhanced photosynthesis efficiency, and gas exchange rates of photosynthesis were 1.3- and 1.2-fold higher for pflp-1 and pflp-2 than WT respectively. Significantly higher electron transport rates of pflp rice plants were observed. Moreover, photosynthetic products, such as fructose, glucose, sucrose and starch contents of pflp transgenic lines were increased accordingly. Molecular evidences of carbohydrate metabolism related genes activities (osHXK5, osHXK6, osAGPL3, osAGPS2α, osSPS, ospFBPase, oscFBPase, and osSBPase) in transgenic lines were higher than those of WT. For performance of crop production, 1000-grain weight for pflp-1 and pflp-2 rice plants were 52.9 and 41.1 g that were both significantly higher than 31.6 g for WT, and panicles weights were 1.4- and 1.2-fold higher than WT. Panicle number, tiller number per plants for pflp rice plants were all significantly higher compared with those of WT where there was no significant difference observed between two pflp rice plants. Taken altogether; this study demonstrated that constitutive pflp expression can improve rice production by enhancing the capacity of photosynthetic carbon assimilation.

Ectopically expressed sweet pepper ferredoxin PFLP enhances disease resistance to Pectobacterium carotovorum subsp. carotovorum affected by harpin and protease-mediated hypersensitive response in Arabidopsis.

Plant ferredoxin-like protein (PFLP) is a photosynthesis-type ferredoxin (Fd) found in sweet pepper. It contains an iron-sulphur cluster that receives and delivers electrons between enzymes involved in many fundamental metabolic processes. It has been demonstrated that transgenic plants overexpressing PFLP show a high resistance to many bacterial pathogens, although the mechanism remains unclear. In this investigation, the PFLP gene was transferred into Arabidopsis and its defective derivatives, such as npr1 (nonexpresser of pathogenesis-related gene 1) and eds1 (enhanced disease susceptibility 1) mutants and NAHG-transgenic plants. These transgenic plants were then infected with the soft-rot bacterial pathogen Pectobacterium carotovorum subsp. carotovorum (Erwinia carotovora ssp. carotovora, ECC) to investigate the mechanism behind PFLP-mediated resistance. The results revealed that, instead of showing soft-rot symptoms, ECC activated hypersensitive response (HR)-associated events, such as the accumulation of hydrogen peroxide (H2 O2 ), electrical conductivity leakage and expression of the HR marker genes (ATHSR2 and ATHSR3) in PFLP-transgenic Arabidopsis. This PFLP-mediated resistance could be abolished by inhibitors, such as diphenylene iodonium (DPI), 1-l-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane (E64) and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), but not by myriocin and fumonisin. The PFLP-transgenic plants were resistant to ECC, but not to its harpin mutant strain ECCAC5082. In the npr1 mutant and NAHG-transgenic Arabidopsis, but not in the eds1 mutant, overexpression of the PFLP gene increased resistance to ECC. Based on these results, we suggest that transgenic Arabidopsis contains high levels of ectopic PFLP; this may lead to the recognition of the harpin and to the activation of the HR and other resistance mechanisms, and is dependent on the protease-mediated pathway.

Kelsoenethiol and dikelsoenyl ether, two unique kelsoane-type sesquiterpenes, from the Formosan soft coral Nephthea erecta.

Two new kelsoane-type sesquiterpenes, namely kelsoenethiol (1) and dikelsoenyl ether (2), were obtained from the Formosan soft coral Nephthea erecta. Their structures were elucidated through extensive spectroscopic analyses, ESI orbitrap mass and quantum chemical calculations (QCC). The cytotoxicity against A-459 (human lung carcinoma), P-388 (mouse lymphocytic leukemia), and HT-29 (human colon adenocarcinoma) cancer cell lines of 1 and 2 was evaluated in vitro. Compound 1 showed cytotoxicity against P-388 and HT-29 cells with ED50s of 1.3 and 1.8 µg/mL, respectively.

Downregulation of putative UDP-glucose: flavonoid 3-O-glucosyltransferase gene alters flower coloring in Phalaenopsis.

Anthocyanin is the primary pigment contributing to red, violet, and blue flower color formation. The solubility of anthocyanins is enhanced by UDP glucose: flavonoid 3-O-glucosyltransferase (UFGT) through transfer of the glucosyl moiety from UDP-glucose to 3-hydroxyl group to produce the first stable pigments. To assess the possibility that UFGT is involved in the flower color formation in Phalaenopsis, the transcriptional activities of PeUFGT3, and other flower color-related genes in developing red or white flower buds were examined using RT-PCR analysis. In contrast with chalcone synthase, chalcone isomerase, and anthocyanidin synthase genes, PeUFGT3 transcriptional activity was higher expressed in the red color of Phalaenopsis cultivars. In the red labellum of Phalaenopsis 'Luchia Lady', PeUFGT3 also showed higher expression levels than that in the white perianth. PeUFGT3 was predominantly expressed in the red region of flower among various Phalaenopsis cultivars. To investigate the role of PeUFGT3 in red flower color formation, PeUFGT3 was specifically knocked down using RNA interference technology via virus inducing gene silencing in Phalaenopsis. The PeUFGT3-suppressed Phalaenopsis exhibited various levels of flower color fading that was well correlated with the extent of reduced level of PeUFGT3 transcriptional activity. Furthermore, there was a significant decrease in anthocyanin content in the PeUFGT3-suppressed Phalaenopsis flowers. The decrease of anthocyanin content due to PeUFGT3 gene silencing possibly caused the faded flower color in PeUFGT3-suppressed Phalaenopsis. Consequently, these results suggested that the glycosylation-related gene PeUFGT3 plays a critical role in red color formation in Phalaenopsis.

Ectopic ferredoxin I protein promotes root hair growth through induction of reactive oxygen species in Arabidopsis thaliana.

Ferredoxin I (Fd-1) is a protein existing in green tissues as an electron carrier for photosynthesis. Reactive oxygen species (ROS) are generated from an over-accumulation of electrons in photosynthetic electron chains. In previous studies, plant ferredoxin-like protein (PFLP) transgenic plants could be made resistant to virulent pathogens, by inducing the generation of ROS. The generation of ROS is closely associated with root hair development, increasing with the elongation of root hairs. We propose that an ectopic expression of pflp may alter root hair development through the enhanced generation of ROS. In this report, Arabidopsis transformed with pflp was generated to determine the potential role of PFLP in root development. Transgenic Arabidopsis exhibited longer root hairs with a significant increase in endogenous H(2)O(2) compared with wild type. The growth of transgenic lines in root hairs was inhibited when treated with NADPH oxidase inhibitor. Results suggest that an over-expression of pflp had enhanced the accumulation of H(2)O(2) in the roots and further promoted the growth of root hairs. Transcriptional activities of root hair development-related and redox-regulated genes were mediated through increased levels of ROS, to alter the growth of transgenic lines in root hairs. In summary, we propose that an ectopic expression of pflp promotes root hair growth, resulting from an enhancement of ROS production.

Plant ferredoxin-like protein (PFLP) outside chloroplast in Arabidopsis enhances disease resistance against bacterial pathogens.

Protection of crops against bacterial disease is an important issue in agricultural production. One of the strategies to lead plants become resistant against bacterial pathogens is employing a transgene, like plant ferredoxin-like protein (PFLP). PFLP is a photosynthetic type ferredoxin isolated from sweet pepper and contains a signal peptide for targeting towards chloroplasts. Our previous reports indicated that transgenic plants with this protein are more resistant against bacterial pathogens. However, this heterologous protein was visualized not only inside the chloroplasts, but also in the cytoplasm. In this article, we moved to study its heterologous expression in Arabidopsis by expressing the protein in chloroplast, apoplast and cytoplasm. This work was achieved by engineering a chloroplast target (CPF), an apoplast target (ESF), and cytoplasm target (DF) plants. The expression and subcellular localization of PFLP were analyzed by Western blot and immuno-staining by confocal microscopy, respectively. We tested the ability of the transgenic Arabidopsis for resistance to two Ralstonia solanacearum strains and their ability to increase the hypersensitive response (HR) triggered by harpin (HrpZ) from Pseudomonas syringae. The DF and ESF plants conferred resistance against bacterial wilt strains and increased HR by harpin, but no resistance found in the CPF plants. In addition, we determined the level of reduced ascorbate in all transgenic plants and further analyzed the expression of two NADPH-oxidase genes (AtrbohD and AtrbohF) in ESF plant. Among the transgenic Arabidopsis plants, ESF plants confer the highest resistance to bacterial pathogens and followed by DF plants. We concluded that PFLP enhances disease resistance in Arabidopsis when expressed in the apoplast or in cytoplasm but not when targeted into the chloroplast. This study provides a strategy for molecular breeding to improve resistance of crops against bacterial pathogens.

Cool-night temperature induces spike emergence and affects photosynthetic efficiency and metabolizable carbohydrate and organic acid pools in Phalaenopsis aphrodite.

After being acclimated to constant warm (28 degrees C day/28 degrees C night) and cool-night temperature (28 degrees C day/20 degrees C night) regimes in growth chambers for 2 weeks, the two groups of mature Phalaenopsis aphrodite subsp. formosana plants both clearly exhibited a diurnal oscillation of stomatal conductance, net CO(2) uptake rate, malate and starch levels, and the phosphoenolpyruvate carboxylase (EC 4.1.1.31) and NAD(+)-malic enzyme (EC 1.1.1.39) activities. Hence, P. aphrodite is an obligate crassulacean acid metabolism plant. Nevertheless, different night temperature greatly affected both the stomatal conductance and the contribution of ambient and respiratory CO(2) to the nocturnal accumulation of malate. However, the amounts of nocturnal accumulated malate and daily deposited starch appeared to have no significant difference between the two groups. These results demonstrate that P. ahrodite is congruent with the characteristics of CAM plants having great flexibility and plasticity in response to changes in environmental conditions. In addition, the formation of reproductive stem, viz. spike, was noticeably inhibited by a constant warm temperature, but induced by a fluctuating warm day and cool night condition. The relationship between the metabolic pool variation and spike induction of Phalaenopsis is also discussed.

Production of soft rot resistant calla lily by expressing a ferredoxin-like protein gene (pflp) in transgenic plants.

An efficient protocol for the Agrobacterium tumefaciens-mediated transformation of calla lily (Zantedeschia elliottiana (W. Wats.) Engl. cultivar 'Florex Gold') is described. Shoot basal discs were co-cultivated with A. tumefaciens C58C1 carrying a plasmid containing neomycin phosphotransferase (nptII) and plant ferredoxin-like protein (pflp) genes. After Agrobacterium co-cultivation, the shoot basal discs were exposed to 100 mg l(-1) kanamycin for selection. Twenty-eight out of 260 discs (10.8%) were found to have survived and produced shoot clusters. Twenty-six of these were confirmed to contain the pflp transgene by PCR, ending up in 10% transformation efficiency. The disease resistance investigation revealed that 18 transgenic plants exhibited resistance to soft rot disease caused by Erwinia carotovora subsp. carotovora. The presence of pflp gene was demonstrated by PCR, and its accumulation and activity was confirmed by Western blot and disease resistance assay. This was the first report to show the successful transformation and resistance to a bacterial pathogen in Zantedeschia. The protocol is useful for the quality improvement of calla lily through genetic transformation.

Disease resistance to bacterial pathogens affected by the amount of ferredoxin-I protein in plants.

SUMMARY Ferredoxin-I (Fd-I) is a fundamental protein that is involved in several metabolic pathways. The amount of Fd-I found in plants is generally regulated by environmental stress, including biotic and abiotic events. In this study, the correlation between quantity of Fd-I and plant disease resistance was investigated. Fd-I levels were increased by inoculation with Pseudomonas syringae pv. syringae but were reduced by Erwinia carotovora ssp. carotovora. Transgenic tobacco over-expressing Fd-I with the sense sweet pepper Fd-I gene (pflp) was resistant to E. carotovora ssp. carotovora and the saprophytic bacterium P. fluorescens. By contrast, transgenic tobacco with reduced total Fd-I and the antisense pflp gene was susceptible to E. carotovora ssp. carotovora and P. fluorescens. Both of these transgenic tobaccos were resistant to P. syringae pv. syringae. By contrast, the mutated E. carotovora ssp. carotovora, with a defective harpin protein, was able to invade the sense-pflp transgenic tobacco as well as the non-transgenic tobacco. An in vitro kinase assay revealed that harpin could activate unidentified kinases to phosphorylate PFLP. These results demonstrate that Fd-I plays an important role in the disease defence mechanism.

Resistance enhancement of transgenic tomato to bacterial pathogens by the heterologous expression of sweet pepper ferredoxin-I protein.

ABSTRACT Expression of a foreign gene to enhance plant disease resistance to bacterial pathogens is a favorable strategy. It has been demonstrated that expressing sweet pepper ferredoxin-I protein (PFLP) in transgenic plants can enhance disease resistance to bacterial pathogens that infect leaf tissue. In this study, PFLP was applied to protect tomato (Lycopersicon esculentum cv. cherry Cln1558a) from the root-infecting pathogen, Ralstonia solanacearum. Independent R. solanacearum resistant T(1) lines were selected and bred to produce homozygous T(2) generations. Selected T(2) transgenic lines 24-18-7 and 26-2-1a, which showed high expression levels of PFLP in root tissue, were resistant to disease caused by R. solanacearum. In contrast, the transgenic line 23-17-1b and nontransgenic tomato, which showed low expression levels of PFLP in root tissue, were not resistant to R. solanacearum infection. The expansion of R. solanacearum populations in stem tissue of transgenic tomato line 24-18-7 was limited compared with the nontransgenic tomato Cln1558a. Using a detached leaf assay, transgenic line 24-18-7 was also resistant to maceration caused by E. carotovora subsp. carotovora; however, resistance to E. carotovora subsp. carotovora was less apparent in transgenic lines 26-2-1a and 23-17-1b. These results demonstrate that PFLP is able to enhance disease resistance at different levels to bacterial pathogens in individual tissue of transgenic tomato.

Expression of the hypersensitive response-assisting protein in Arabidopsis results in harpin-dependent hypersensitive cell death in response to Erwinia carotovora.

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin(Pss)-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H(2)O(2) and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN(-) mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.

Ferredoxin from sweet pepper (Capsicum annuum L.) intensifying harpin(pss)-mediated hypersensitive response shows an enhanced production of active oxygen species (AOS).

The hypersensitive response (HR) is a form of cell death associated with plant resistance to pathogen infection. Harpin(pss), an elicitor from the bacterium Pseudomonas syringae pv. syringae, induces a HR in non-host plants. Previously, we reported an amphipathic protein from sweet pepper interfering with harpin(pss)-mediated HR. In this report, we isolated and characterized a cDNA clone encoded that amphipathic protein from sweet pepper. This protein is designated as PFLP (plant ferredoxin-like protein) by virtue of its high homology with plant ferredoxin protein containing an N-terminal signal peptide responsible for chloroplast targeting and a putative 2Fe-2S domain responsible for redox activity. Recombinant PFLP obtained from Escherichia coli was able to significantly increase active oxygen species (AOS) generation when mixed with harpin(pss) in tobacco suspension cells. It also showed enhanced HR when co-infiltrated with harpin(pss) in tobacco leaves. We used a transgenic tobacco suspension cells system that constitutively expresses the Pflp gene driven by the CaMV 35S promoter to study the function of PFLP in enhancing harpin(pss)-mediated hypersensitive cell death in vivo. In response to harpin(pss), suspension cells derived from Pflp transgenic tobacco showed a significant increase both in the generation of AOS and in cell death as compared to the wild type. AOS inhibitors diphenylene iodonium chloride (DPI) and lanthanum chlorate (LaCl3) were used to study the involvement of AOS in harpin(pss)-induced cell death. Our results demonstrate enhanced generation of AOS is necessary to cause enhanced hypersensitive cell death in Pflp transgenic tobacco cells and it is plasma membrane-bound NADPH-oxidase-dependent. Sub-cellular localization studies showed that PFLP is present in the cytoplasm and chloroplast of Pflp transgenic tobacco cells, but only in the chloroplast, not in the cytoplasm, of wild-type tobacco cells. It is possible that PFLP can change the redox state of the cell upon harpin(pss) inoculation to increase AOS generation and hypersensitive cell death. Overall, this study will provide a new insight in the functional properties of ferredoxin in hypersensitive cell death.

Constitutive expression of hrap gene in transgenic tobacco plant enhances resistance against virulent bacterial pathogens by induction of a hypersensitive response.

Hypersensitive response-assisting protein (HRAP) has been previously reported as an amphipathic plant protein isolated from sweet pepper that intensifies the harpin(Pss)-mediated hypersensitive response (HR). The hrap gene has no appreciable similarity to any other known sequences, and its activity can be rapidly induced by incompatible pathogen infection. To assess the function of the hrap gene in plant disease resistance, the CaMV 35S promoter was used to express sweet pepper hrap in transgenic tobacco. Compared with wild-type tobacco, transgenic tobacco plants exhibit more sensitivity to harpin(Pss) and show resistance to virulent pathogens (Pseudomonas syringae pv. tabaci and Erwinia carotovora subsp. carotovora). This disease resistance of transgenic tobacco does not originate from a constitutive HR, because endogenous level of salicylic acid and hsr203J mRNA showed similarities in transgenic and wildtype tobacco under noninfected conditions. However, following a virulent pathogen infection in hrap transgenic tobacco, hsr203J was rapidly induced and a micro-HR necrosis was visualized by trypan blue staining in the infiltration area. Consequently, we suggest that the disease resistance of transgenic plants may result from the induction of a HR by a virulent pathogen infection.