Expression of plant ferredoxin-like protein (PFLP) enhances tolerance to heat stress in Arabidopsis thaliana.

Under adverse environments, plants produce reactive oxygen species (ROS), which can trigger cell death when their accumulation surpasses the antioxidant capacity of ROS scavenging systems. These systems function in chloroplasts mainly through the ascorbate-mediated water-water cycle, in which ascorbate is photoreduced by ferredoxin in the photosynthetic system. Our previous study showed that the fraction of the reduced form of ascorbate was increased in ferredoxin-transgenic Arabidopsis (CPF) plants which overexpressed plant ferredoxin-like protein (PFLP) in their chloroplasts. Thus, we hypothesized that expression of PFLP could alter the tolerance of plants to abiotic stresses through increasing reduced form of ascorbate. In this study, we found that two CPF lines exhibited lower mortality rates at five days, following two days of heat treatment. Compared to non-transgenic wild type (Col-0) plants, CPF plants exhibited decreased H2O2 content, MDA accumulation, and ion leakage after heat treatment. To confirm the efficacy of ferredoxin against heat stress in chloroplasts, we evaluated two RNA interference (RNAi) lines on two endogenous ferredoxin isoforms, Atfd1 or Atfd2, of Arabidopsis plants. Both lines not only decreased their amounts of ascorbate, but also exhibited adverse reactions following heat treatment. Based on these results, we conclude that expression of PFLP in chloroplasts can confer tolerance to heat stress. This tolerance might be associated with the increasing of ascorbate in plants.

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.

Overexpression of ferredoxin, PETF, enhances tolerance to heat stress in Chlamydomonas reinhardtii.

Reactive oxygen species (ROS) produced by plants in adverse environments can cause damage to organelles and trigger cell death. Removal of excess ROS can be achieved through the ascorbate scavenger pathway to prevent plant cell death. The amount of this scavenger can be regulated by ferredoxin (FDX). Chloroplastic FDXs are electron transfer proteins that perform in distributing photosynthetic reducing power. In this study, we demonstrate that overexpression of the endogenous photosynthetic FDX gene, PETF, in Chlamydomonas reinhardtii could raise the level of reduced ascorbate and diminish H2O2 levels under normal growth conditions. Furthermore, the overexpressing PETF transgenic Chlamydomonas lines produced low levels of H2O2 and exhibited protective effects that were observed through decreased chlorophyll degradation and increased cell survival under heat-stress conditions. The findings of this study suggest that overexpression of PETF can increase the efficiency of ROS scavenging in chloroplasts to confer heat tolerance. The roles of PETF in the downregulation of the ROS level offer a method for potentially improving the tolerance of crops against heat stress.

Knockdown of PsbO leads to induction of HydA and production of photobiological H2 in the green alga Chlorella sp. DT.

Green algae are able to convert solar energy to H2 via the photosynthetic electron transport pathway under certain conditions. Algal hydrogenase (HydA, encoded by HYDA) is in charge of catalyzing the reaction: 2H(+)+2e(-)↔H2 but usually inhibited by O2, a byproduct of photosynthesis. The aim of this study was to knockdown PsbO (encoded by psbO), a subunit concerned with O2 evolution, so that it would lead to HydA induction. The alga, Chlorella sp. DT, was then transformed with short interference RNA antisense-psbO (siRNA-psbO) fragments. The algal mutants were selected by checking for the existence of siRNA-psbO fragments in their genomes and the low amount of PsbO proteins. The HYDA transcription and the HydA expression were observed in the PsbO-knockdown mutants. Under semi-aerobic condition, PsbO-knockdown mutants could photobiologically produce H2 which increased by as much as 10-fold in comparison to the wild type.

C-terminal region of plant ferredoxin-like protein is required to enhance resistance to bacterial disease in Arabidopsis thaliana.

Protein phosphorylation is an important biological process associated with elicitor-induced defense responses in plants. In a previous report, we described how plant ferredoxin-like protein (PFLP) in transgenic plants enhances resistance to bacterial pathogens associated with the hypersensitive response (HR). PFLP possesses a putative casein kinase II phosphorylation (CK2P) site at the C-terminal in which phosphorylation occurs rapidly during defense response. However, the contribution of this site to the enhancement of disease resistance and the intensity of HR has not been clearly demonstrated. In this study, we generated two versions of truncated PFLP, PEC (extant CK2P site) and PDC (deleted CK2P site), and assessed their ability to trigger HR through harpin (HrpZ) derived from Pseudomonas syringae as well as their resistance to Ralstonia solanacearum. In an infiltration assay of HrpZ, PEC intensified harpin-mediated HR; however, PDC negated this effect. Transgenic plants expressing these versions indicate that nonphosphorylated PFLP loses its ability to induce HR or enhance disease resistance against R. solanacearum. Interestingly, the CK2P site of PFLP is required to induce the expression of the NADPH oxidase gene, AtrbohD, which is a reactive oxygen species producing enzyme. This was further confirmed by evaluating the HR on NADPH oxidase in mutants of Arabidopsis. As a result, we have concluded that the CK2P site is required for the phosphorylation of PFLP to enhance disease resistance.

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.

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.

The sweet pepper ferredoxin-like protein (pflp) conferred resistance against soft rot disease in Oncidium orchid.

Genetic engineering to date has not been used to introduce disease resistance genes into the orchid gene pool. The ferredoxin-like protein gene originally isolated from sweet pepper is thought to function as a natural defense against infection due to its antimicrobial properties. Hence it was reasoned that introduction of this gene might produce Oncidium plants resistant to Erwinia carotovora, the causal agent for the soft rot disease. An expression vector containing sweet pepper ferredoxin-like protein (pflp) cDNA, hph and gusA coding sequence was successfully transformed into protocorm-like bodies (PLBs) of Oncidium orchid, using Agrobacterium tumefaciens strain EHA105. A total of 17 independent transgenic orchid lines was obtained, out of which six transgenic lines (beta-glucuronidase (GUS) positive) were randomly selected and confirmed by Southern, northern and western blot analyses. A bioassay was conducted on the transgenic lines. Transgenic plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest that pflp may be an extremely useful gene for genetic engineering strategies in orchids to confer resistance against soft rot disease.

Sweet pepper ferredoxin-like protein ( pflp) gene as a novel selection marker for orchid transformation.

A novel method for selection of transgenic plants utilizing the sweet pepper ( Capsicum annuum L.) ferredoxin-like protein ( pflp) gene as selection marker and Erwinia carotovora as the selection agent has been developed. An expression vector containing a pflp cDNA driven by a cauliflower mosaic virus 35S promoter was successfully transformed into protocorm-like bodies of Oncidium orchid by Agrobacterium tumefaciens and particle bombardment, respectively. Erwinia carotovora was used as a selection agent to screen transformants, thereby obtaining transgenic plants without the use of an antibiotic selection agent. A total of 32 independent transgenic orchid lines were obtained, out of which 9 transgenic lines (beta-glucuronidase positive) were randomly selected and confirmed by Southern and northern blot analyses. The transgenic orchid plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest the novel use of the pflp gene as a resistance selection marker in plant genetic engineering strategies. In the future, the use of the pflp gene as a selection marker may facilitate the use of smaller gene constructs due to removal of bulky antibiotic selection and reporter genes. These constructs can then be used to incorporate additional genes of choice.

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.