Ferredoxin-like Protein Research Articles

S-adenosyl-L-methionine is the unexpected methyl donor for the methylation of mercury by the membrane-associated HgcAB complex

Mercury (Hg) is a heavy metal that exhibits high biological toxicity. Monomethylmercury and dimethylmercury are neurotoxins and a significant environmental concern as they bioaccumulate and biomagnify within the aquatic food web. Microbial Hg methylation involves two proteins, HgcA and HgcB. Here, we show that HgcA and HgcB can be heterologously coexpressed, and the HgcAB complex can be purified. We demonstrated that HgcA is a membrane-associated cobalamin-dependent methyltransferase and HgcB is a ferredoxin-like protein containing two [4Fe-4S] clusters. Further, spectroscopic and kinetic results demonstrate that S-adenosyl-L-methionine (SAM) donates the methyl group to Hg in a two-step reaction involving a methylcob(III)alamin intermediate including Co-thiolate ligation from a conserved Cys residue. Our findings uncover a biological role for SAM in microbial Hg methylation.

Improving photosynthetic efficiency of rice via over-expressing a ferredoxin-like protein gene from Methanothermobacter thermautotrophicus.

Ferredoxins (Fds) are crucial in various essential plant metabolic processes, including photosynthesis, fermentation and aerobic nitrogen fixation, due to their role in electron transport rate (ETR). However, the full scope of ferredoxin's function across prokaryotes and eukaryotic plants remains less understood. This study investigated the effect of MtFd from Methanothermobacter thermoautotrophicus on rice photosynthetic efficiency. We found that MtFd was localized in the chloroplasts of rice protoplasts. Transgenic analysis showed that MtFd significantly enhanced the photosynthetic capacity compared to the wild-type plants. This enhancement was evident through increased ETR, NADPH content and net photosynthetic rates, as well as decreased non-photochemical quenching (NPQ). Despite similar biomass to wild type plants, MtFd transgenic plants exhibited a marked increase in grain size and the 1000-grian weight. The elevated ETR and surplus free electrons in transgenic plants result in a considerable rise in cellular ROS content, which in turn enhances the enzymatic activity of the antioxidant system. In summary, our findings suggest that introducing the Fd protein from M. thermoautotrophicus into transgenic rice improves photosynthetic efficiency by accelerating ETR, which triggers the cellular oxidative stress response.

Identifying and Characterizing Candidate Genes Contributing to a Grain Yield QTL in Wheat.

The current study focuses on identifying the candidate genes of a grain yield QTL from a double haploid population, Westonia × Kauz. The QTL region spans 20 Mbp on the IWGSC whole-genome sequence flank with 90K SNP markers. The IWGSC gene annotation revealed 16 high-confidence genes and 41 low-confidence genes. Bioinformatic approaches, including functional gene annotation, ontology investigation, pathway exploration, and gene network study using publicly available gene expression data, enabled the short-listing of four genes for further confirmation. Complete sequencing of those four genes demonstrated that only two genes are polymorphic between the parental cultivars, which are the ferredoxin-like protein gene and the tetratricopeptide-repeat (TPR) protein gene. The two genes were selected for downstream investigation. Two SNP variations were observed in the exon for both genes, with one SNP resulting in changes in amino acid sequence. qPCR-based gene expression showed that both genes were highly expressed in the high-yielding double haploid lines along with the parental cultivar Westonia. In contrast, their expression was significantly lower in the low-yielding lines in the other parent. It can be concluded that these two genes are the contributing genes to the grain yield QTL.

Structural and biophysical properties of a [4Fe[sbnd]4S] ferredoxin-like protein from Synechocystis sp. PCC 6803 with a unique two domain structure

Electron carrier proteins (ECPs), binding iron‐sulfur clusters, are vital components within the intricate network of metabolic and photosynthetic reactions. They play a crucial role in the distribution of reducing equivalents. In Synechocystis sp. PCC 6803, the ECP network includes at least nine ferredoxins. Previous research, including global expression analyses and protein binding studies, has offered initial insights into the functional roles of individual ferredoxins within this network. This study primarily focuses on Ferredoxin 9 (slr2059). Through sequence analysis and computational modeling, Ferredoxin 9 emerges as a unique ECP with a distinctive two-domain architecture. It consists of a C-terminal iron‑sulfur binding domain and an N-terminal domain with homology to Nil-domain proteins, connected by a structurally rigid 4-amino acid linker. Notably, in contrast to canonical [2Fe2S] ferredoxins exemplified by PetF (ssl0020), which feature highly acidic surfaces facilitating electron transfer with photosystem I reaction centers, models of Ferredoxin 9 reveal a more neutral to basic protein surface. Using a combination of electron paramagnetic resonance spectroscopy and square-wave voltammetry on heterologously produced Ferredoxin 9, this study demonstrates that the protein coordinates 2×[4Fe4S]2+/1+ redox-active and magnetically interacting clusters, with measured redox potentials of −420 ± 9 mV and − 516 ± 10 mV vs SHE. A more in-depth analysis of Fdx9's unique structure and protein sequence suggests that this type of Nil-2[4Fe4S] multi-domain ferredoxin is well conserved in cyanobacteria, bearing structural similarities to proteins involved in homocysteine synthesis in methanogens.

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 4pm and 10pm 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.

Characterization of ferredoxins from the thermophilic, acetogenic bacterium Thermoanaerobacter kivui.

A major electron carrier involved in energy and carbon metabolism in the acetogenic model organism Thermoanaerobacter kivui is ferredoxin, an iron-sulfur-containing, electron-transferring protein. Here, we show that the genome of T. kivui encodes four putative ferredoxin-like proteins (TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530). All four genes were cloned, a His-tag encoding sequence was added and the proteins were produced from a plasmid in T. kivui. The purified proteins had an absorption peak at 430 nm typical for ferredoxins. The determined iron-sulfur content is consistent with the presence of two predicted [4Fe4S] clusters in TKV_c09620 and TKV_c19530 or one predicted [4Fe4S] cluster in TKV_c16450 and TKV_c10420 respectively. The reduction potential (Em ) for TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530 wasdetermined to be -386 ± 4 mV, -386 ± 2 mV, -559 ± 10 mV and -557 ± 3 mV, respectively. TKV_c09620 and TKV_c16450 served as electron carriers for different oxidoreductases from T. kivui. Deletion of the ferredoxin genes led to only a slight reduction of growth on pyruvate or autotrophically on H2 + CO2 . Transcriptional analysis revealed that TKV_c09620 was upregulated in a ΔTKV_c16450 mutant and vice versa TKV_c16450 in a ΔTKV_c09620 mutant, indicating that TKV_c09620 and TKV_c16450 can replace each other. In sum, our data are consistent with the hypothesis that TKV_c09620 and TKV_c16450 are ferredoxins involved in autotrophic and heterotrophic metabolism of T. kivui.

Structural And Functional Relationship Study In Plant Salinity Stress

In today’s world the increased use of low-quality water for irrigation and soil salinization, saltness is a huge biotic problem that effecting factory growth and product in numerous corridors in the world. In repercussion to salt stress, plant cells accumulate compatible solutes and redistribute ions, all of which contribute them to acclimate to a low soil water potential. In addition, the endogenous abscisic acid (ABA) content increases daily, followed by changes in principle genetic expression in salt stress conditions. Here we are going to discuss about the proteins involved in plant salinity stress and their interactions with the salt tolerant factor which has a negative impact with the plant growth system. Some highly evaluated proteins that were observed under this stress are Plant ferredoxin-like protein (PFLP), Arabidopsis thaliana Salt Tolerance Related Protein (STRP), OsEXPA7, ribulose-1, 5-bisphosphate carboxylase (RuBP), etc. This report is also going to focus on various transcription factors (tf), responsible for the enhanced tolerance under biological activity of plant development. TFs bZIP, MYBs, bHLH, C2H2-Dof, G2-like, AP2-EREBP, NAC, GATA, ARF, ERF, bZIP and MYB which were highly up- regulated in salt stress response.

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.

Using Dynamic Changes of Chlorophyll Fluorescence in Arabidopsis thaliana to Evaluate Plant Immunity-Intensifying Bacillus spp. Strains.

The integral defense responses of plants triggered by the small molecules of plant pathogens are regarded as plant immunity. The pathogen-associated molecular pattern-triggered immunity (PTI) occurs on the recognition of a pathogen by receptors on plant cell surfaces as an infection begins. During the activation of PTI, the effectiveness of a plant's photosynthetic system may be altered. In this study, chlorophyll fluorescence was used to assay the dynamic changes of PTI. When we used flg22Pst as an elicitor, we found that the photosynthetic electron transport rate (ETR) of Arabidopsis thaliana Col-0 was significantly decreased at 2, 4, and 24 h on treatment with a PTI-intensifying protein, plant ferredoxin-like protein (PFLP). In addition, this reduction in the photosynthetic ETR was also carried out with a PTI-intensifying Bacillus amyloliquefaciens strain, PMB05, on the induction of flg22Pst. The disease resistance against bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) was still enhanced by PMB05. Interestingly, among the eight tested Bacillus species strains, the PTI triggered by HrpNPcc from P. carotovorum subsp. carotovorum exhibited an ETR that was significantly decreased by PMB05. Furthermore, this decrease was consistent with rapid H2O2 generation and callose deposition triggered by HrpNPcc and the disease resistance against bacterial soft rot. Taken together, such results led us to conclude that the assay based on the ETR established in this study can be used as a model for evaluating the effectiveness of plant immunity-intensifying microbes for controlling plant diseases.

The Ferredoxin-Like Protein FerR Regulates PrbP Activity in Liberibacter asiaticus.

In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that regulates gene expression through interactions with the RNA polymerase β-subunit and a specific sequence on the promoter region. The constitutive expression of prbP observed upon chemical inactivation of PrbP-DNA interactions in vivo indicated that the expression of prbP was not autoregulated at the level of transcription. This observation suggested that a modulatory mechanism via protein-protein interactions may be involved. In silico genome association analysis identified FerR (CLIBASIA_01505), a putative ferredoxin-like protein, as a PrbP-interacting protein. Using a bacterial two-hybrid system and immunoprecipitation assays, interactions between PrbP and FerR were confirmed. In vitro transcription assays were used to show that FerR can increase the activity of PrbP by 16-fold when present in the PrbP-RNA polymerase reaction mixture. The FerR protein-protein interaction surface was predicted by structural modeling and followed by site-directed mutagenesis. Amino acids V20, V23, and C40 were identified as the most important residues in FerR involved in the modulation of PrbP activity in vitro The regulatory mechanism of FerR abundance was examined at the transcription level. In contrast to prbP of L. asiaticus (prbPLas), mRNA levels of ferR of L. asiaticus (ferRLas) are induced by an increase in osmotic pressure. The results of this study revealed that the activity of the transcriptional activator PrbPLas is modulated via interactions with FerRLas The induction of ferRLas expression by osmolarity provides insight into the mechanisms of adjusting gene expression in response to host environmental signals in L. asiaticusIMPORTANCE The rapid spread and aggressive progression of huanglongbing (HLB) in the major citrus-producing areas have raised global recognition of and vigilance to this disease. As a result, the causative agent, Liberibacter asiaticus, has been investigated from various perspectives. However, gene expression regulatory mechanisms that are important for the survival and persistence of this intracellular pathogen remain largely unexplored. PrbP is a transcriptional accessory protein important for L. asiaticus survival in the plant host. In this study, we investigated the interactions between PrbP in L. asiaticus (PrbPLas) and a ferredoxin-like protein (FerR) in L. asiaticus, FerRLas We show that the presence of FerR stabilizes and augments the activity of PrbPLas In addition, we demonstrate that the expression of ferR is induced by increases in osmolarity in Liberibacter crescens Altogether, these results suggest that FerRLas and PrbPLas may play important roles in the regulation of gene expression in response to changing environmental signals during L. asiaticus infection in the citrus host.

PFLP-Intensified Disease Resistance Against Bacterial Soft Rot Through the MAPK Pathway in PAMP-Triggered Immunity

Bacterial soft rot is a devastating disease affecting a variety of vegetable crops worldwide. One strategy for controlling this disease could be the ectopic expression of the plant ferredoxin-like protein (pflp) gene. PFLP was previously shown to intensify pathogen-associated molecular pattern-triggered immunity (PTI), an immune response triggered, for example, by the flagellin epitope flg22. To gain further insight into how PFLP intensifies PTI, flg22 was used as an elicitor in Arabidopsis thaliana. First, PFLP was confirmed to intensify the rapid generation of H2O2, callose deposition, and the hypersensitive response when coinfiltrated with flg22. This response correlated with increased expression of the FLG22-induced receptor kinase 1 gene, which is part of the mitogen-activated protein kinase (MAPK) pathway. Although the increased response to flg22 alone did not depend on the MAPK pathway genes MEKK1, MKK5, and MPK6, the protective effect of PFLP decreased when plants mutated in these genes were inoculated with Pectobacterium carotovorum subsp. carotovorum. Furthermore, expression of PR1 and PDF1.2 also increased upon treatment with flg22 in the presence of PFLP. Taken together, these results suggest that activation of the MAPK pathway contributes to the increased resistance to bacterial soft rot observed in plants treated with PFLP.

The Ferredoxin-Like Proteins HydN and YsaA Enhance Redox Dye-Linked Activity of the Formate Dehydrogenase H Component of the Formate Hydrogenlyase Complex.

Formate dehydrogenase H (FDH-H) and [NiFe]-hydrogenase 3 (Hyd-3) form the catalytic components of the hydrogen-producing formate hydrogenlyase (FHL) complex, which disproportionates formate to H2 and CO2 during mixed acid fermentation in enterobacteria. FHL comprises minimally seven proteins and little is understood about how this complex is assembled. Early studies identified a ferredoxin-like protein, HydN, as being involved in FDH-H assembly into the FHL complex. In order to understand how FDH-H and its small subunit HycB, which is also a ferredoxin-like protein, attach to the FHL complex, the possible roles of HydN and its paralogue, YsaA, in FHL complex stability and assembly were investigated. Deletion of the hycB gene reduced redox dye-mediated FDH-H activity to approximately 10%, abolished FHL-dependent H2-production, and reduced Hyd-3 activity. These data are consistent with HycB being an essential electron transfer component of the FHL complex. The FDH-H activity of the hydN and the ysaA deletion strains was reduced to 59 and 57% of the parental, while the double deletion reduced activity of FDH-H to 28% and the triple deletion with hycB to 1%. Remarkably, and in contrast to the hycB deletion, the absence of HydN and YsaA was without significant effect on FHL-dependent H2-production or total Hyd-3 activity; FDH-H protein levels were also unaltered. This is the first description of a phenotype for the E. coli ysaA deletion strain and identifies it as a novel factor required for optimal redox dye-linked FDH-H activity. A ysaA deletion strain could be complemented for FDH-H activity by hydN and ysaA, but the hydN deletion strain could not be complemented. Introduction of these plasmids did not affect H2 production. Bacterial two-hybrid interactions showed that YsaA, HydN, and HycB interact with each other and with the FDH-H protein. Further novel anaerobic cross-interactions of 10 ferredoxin-like proteins in E. coli were also discovered and described. Together, these data indicate that FDH-H activity measured with the redox dye benzyl viologen is the sum of the FDH-H protein interacting with three independent small subunits and suggest that FDH-H can associate with different redox-protein complexes in the anaerobic cell to supply electrons from formate oxidation.

Exploring the Promiscuity of Phenol Hydroxylase from Pseudomonas stutzeri OX1 for the Biosynthesis of Phenolic Compounds.

Enzyme promiscuity plays an important role in developing biosynthetic pathways for novel target products. Phenol hydroxylase (PH) from Pseudomonas stutzeri OX1 is capable of ortho-hydroxylation of phenol and cresol isomers into counterpart catechols. A small ferredoxin-like protein PHQ was clustered together with the ph gene cluster in the genome of P.stutzeri OX1, and its function was not known. In this study, we found that the existence of PHQ has a promotion effect on the catalytic efficiency of PH. Then, we tested the substrate range of PH using nine different non-natural substrates. We found that PH was a promiscuous hydroxylase that could catalyze ortho-hydroxylation of several non-natural substrates, including catechol, 4-hydroxybenzoic acid and resorcinol. On this basis, linking the catechol biosynthetic pathway with the hydroxylation reaction catalyzed by PH enabled construction of a novel biosynthetic pathway for the synthesis of pyrogallol. This work not only characterized a well-performed PH, but also provided a promising hydroxylation platform for the production of high-value phenolic compounds.

Bioinformatics analysis to assess potential risks of allergenicity and toxicity of HRAP and PFLP proteins in genetically modified bananas resistant to Xanthomonas wilt disease

Banana Xanthomonas wilt (BXW) disease threatens banana production and food security throughout East Africa. Natural resistance is lacking among common cultivars. Genetically modified (GM) bananas resistant to BXW disease were developed by inserting the hypersensitive response-assisting protein (Hrap) or/and the plant ferredoxin-like protein (Pflp) gene(s) from sweet pepper (Capsicum annuum). Several of these GM banana events showed 100% resistance to BXW disease under field conditions in Uganda. The current study evaluated the potential allergenicity and toxicity of the expressed proteins HRAP and PFLP based on evaluation of published information on the history of safe use of the natural source of the proteins as well as established bioinformatics sequence comparison methods to known allergens (www.AllergenOnline.org and NCBI Protein) and toxins (NCBI Protein). The results did not identify potential risks of allergy and toxicity to either HRAP or PFLP proteins expressed in the GM bananas that might suggest potential health risks to humans. We recognize that additional tests including stability of these proteins in pepsin assay, nutrient analysis and possibly an acute rodent toxicity assay may be required by national regulatory authorities.

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 41.1g that were both significantly higher than 31.6g 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.

Structure and Function of a C-C Bond Cleaving Oxygenase in Atypical Angucycline Biosynthesis.

C-C bond ring cleaving oxygenases represent a unique family of enzymes involved in the B ring cleavage reaction only observed in atypical angucycline biosynthesis. B ring cleavage is the key reaction leading to dramatic divergence in the final structures of atypical angucyclines. Here, we present the crystal structure of AlpJ, the first structure of this family of enzymes. AlpJ has been verified as the enzyme catalyzing C-C bond cleavage in kinamycin biosynthesis. The crystal structure of the AlpJ monomer resembles the dimeric structure of ferredoxin-like proteins. The N- and C-terminal halves of AlpJ are homologous, and both contain a putative hydrophobic substrate binding pocket in the "closed" and "open" conformations, respectively. Structural comparison of AlpJ with ActVA-Orf6 and protein-ligand docking analysis suggest that the residues including Asn60, Trp64, and Trp181 are possibly involved in substrate recognition. Site-directed mutagenesis results supported our hypothesis, as mutation of these residues led to nearly a complete loss of the activity of AlpJ. Structural analysis also revealed that AlpJ possesses an intramolecular domain-domain interface, where the residues His50 and Tyr178 form a hydrogen bond that probably stabilizes the three-dimensional structure of AlpJ. Site-directed mutagenesis showed that the two residues, His50 and Tyr178, were vital for the activity of AlpJ. Our findings shed light on the structure and catalytic mechanism of the AlpJ family of oxygenases, which presumably involves two active sites that might function in a cooperative manner.

Expressing stacked HRAP and PFLP genes in transgenic banana has no synergistic effect on resistance to Xanthomonas wilt disease

Banana production in Africa's great lakes region is threatened by the Banana Xanthomonas wilt (BXW) disease caused by Xanthomonas campestris pv. musacearum, a biotrophic pathogen. Transgenic banana plants, cv. “Gonja manjaya,” expressing stacked hypersensitive response-assisting protein gene (HRAP) and the plant ferredoxin-like protein gene (PFLP) were evaluated for resistance against BXW in comparison to transgenic lines having single gene. Transgenic lines with stacked gene as well as single gene had higher resistance to the pathogen than non-transgenic control plants indicated by either no symptom development or delayed symptoms for completely and partially resistant plants, respectively. Transgenic lines also produced more hydrogen peroxide due to pathogen infection and also had higher transcription of stress response genes encoding NPR1, a defense response co-transcriptor, the antimicrobial PR-3 and glutathione S-transferase. However, transcription of PR-1, an indicator for infection with a biotrophic pathogen, was not increased in both stacked and single transgenic lines, indicating a possible shift to infection with a necrotrophic pathogen in plants due to transgenes expression.Expression of stacked HRAP and PFLP genes in transgenic banana lines did not show higher or additive resistance levels against pathogen in comparison to individual genes; however, stacking might provide the benefit of durable resistance in case one transgene function is lost.

Pflp gene transformation in pomegranate (Punica granatum L.) resistance to bacterial blight disease (Xanthomonas axonopodis pv. punicae)

Pomegranate ( Punica granatum L.) is a woody perennial fruit crop grown in arid zones of India, the state of Maharashtra being considered as pomegranate basket contributes to 70 % of the total area followed by Karnataka and Andhra Pradesh. Protection of crops against bacterial disease is an important issue in agricultural production. Pomegranate production in India is severely hampered by the high incidence of bacterial blight disease caused by Xanthomonas axonopodis pv. punicae is air borne, the conventional ways and means of controlling this disease have failed. Evolving a resistant genotype using resistant variety through conventional breeding may be a way out but it is a time consuming process. Transgenic approach appears to be promising to minimize the losses caused by disease. In the present investigation efficient protocols were developed to get healthy and well-formed plants from juvenile and mature-origin explants of the pomegranate cv. ‘Bhagwa’ and transformants with PFLP gene. One of the strategies to lead plants become resistant against bacterial pathogens is employing a transgene, like plant ferredoxin-like protein (PFLP). Different treatment combinations of hormonal concentrations were taken for leaf, petal, nodes and cotyledonary explants to standardize an efficient in vitro regeneration protocol and find out the best treatment for faster regeneration. Agrobacterium tumefaciens carrying gene pCAMBIA construct with the constitutive CaMV35S promoter, PFLP gene, terminator and nptII selectable marker (Kanamycin resistance), was used for transformation of explants. Putative transformants were identified on selection medium containing kanamycin at different concentration. Integration of transgene and expression at various levels were confirmed using PCR. Out of 4 putative transformants analyzed, three plants showed amplification for PFLP gene specific primer. Key words In vitro , Pflp gene, transformation, bacterial blight disease, putative transformants, Agrobacterium tumefaciens

Enhancement of naphthalene tolerance in transgenic Arabidopsis plants overexpressing the ferredoxin-like protein (ADI1) from rice

The ADI1 Arabidopsis plants enhanced tolerance and degradation efficiency to naphthalene and had great potential for phytoremediation of naphthalene in the plant material before composting or harvesting and removal. Naphthalene is a global environmental concern, because this substance is assumed to contribute considerably to human cancer risk. Cleaning up naphthalene contamination in the environment is crucial. Phytoremediation is an efficient technology to clean up contaminants. However, no gene that can efficiently degrade exogenous recalcitrant naphthalene in plants has yet been discovered. Ferredoxin (Fd) is a key player of biological electron transfer reaction in the PAH degradation process. The biochemical pathway for bacterial degradation of naphthalene has been well investigated. In this study, a rice gene, ADI1, which codes for a putative photosynthetic-type Fd, has been transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants enhanced tolerance and degradation efficiency of naphthalene. Compared with wild-type plants, transgenic plants assimilated naphthalene from the culture media faster and removed more of this substance. When taken together, our findings suggest that breeding plants with overexpressed ADI1 gene is an effective strategy to degrade naphthalene in the environment.

Mutation of FdC2 gene encoding a ferredoxin-like protein with C-terminal extension causes yellow-green leaf phenotype in rice

Ferredoxins (Fds) are small iron-sulfur proteins that mediate electron transfer in a wide range of metabolic reactions. Besides Fds, there is a type of Fd-like proteins designated as FdC, which have conserved elements of Fds, but contain a significant C-terminal extension. So far, only two FdC genes of Arabidopsis (Arabidopsis thaliana) have been identified in higher plants and thus the functions of FdC proteins remain largely unknown. In this study, we isolated a yellow-green leaf mutant, 501ys, in rice (Oryza sativa). The mutant exhibited yellow-green leaf phenotype and reduced chlorophyll level. The phenotype of 501ys was caused by mutation of a gene on rice chromosome 3. Map-based cloning of this mutant resulted in identification of OsFdC2 gene (LOC_Os03g48040) showing high identity with Arabidopsis FdC2 gene (AT1G32550). OsFdC2 was expressed most abundantly in leaves and its encoded protein was targeted to the chloroplast. In 501ys mutant, a missense mutation was detected in DNA sequence of the gene, resulting in an amino acid change in the encoded protein. The mutant phenotype was rescued by introduction of the wild-type gene. Therefore, we successfully identified FdC2 gene via map-based cloning approach, and demonstrated that mutation of this gene caused yellow-green leaf phenotype in rice.