PsbN is required for assembly of the photosystem II reaction center in Nicotiana tabacum.

The chloroplast-encoded low molecular weight protein PsbN is annotated as a photosystem II (PSII) subunit. To elucidate the localization and function of PsbN, encoded on the opposite strand to the psbB gene cluster, we raised antibodies and inserted a resistance cassette into PsbN in both directions. Both homoplastomic tobacco (Nicotiana tabacum) mutants psbN-F and psbN-R show essentially the same PSII deficiencies. The mutants are extremely light sensitive and failed to recover from photoinhibition. Although synthesis of PSII proteins was not altered significantly, both mutants accumulated only ∼25% of PSII proteins compared with the wild type. Assembly of PSII precomplexes occurred at normal rates, but heterodimeric PSII reaction centers (RCs) and higher order PSII assemblies were not formed efficiently in the mutants. The psbN-R mutant was complemented by allotopic expression of the PsbN gene fused to the sequence of a chloroplast transit peptide in the nuclear genome. PsbN represents a bitopic trans-membrane peptide localized in stroma lamellae with its highly conserved C terminus exposed to the stroma. Significant amounts of PsbN were already present in dark-grown seedling. Our data prove that PsbN is not a constituent subunit of PSII but is required for repair from photoinhibition and efficient assembly of the PSII RC.

Comparative genomics of the lipid-body-membrane proteins oleosin, caleosin and steroleosin in magnoliophyte, lycophyte and bryophyte.

Lipid bodies store oils in the form of triacylglycerols. Oleosin, caleosin and steroleosin are unique proteins localized on the surface of lipid bodies in seed plants. This study has identified genes encoding lipid body proteins oleosin, caleosin and steroleosin in the genomes of five plants: Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Selaginella moellendorffii and Physcomitrella patens. The protein sequence alignment indicated that each oleosin protein contains a highly-conserved proline knot motif, and proline knob motif is well conserved in steroleosin proteins, while caleosin proteins possess the Dx[D/N]xDG-containing calcium-binding motifs. The identification of motifs (proline knot and knob) and conserved amino acids at active site was further supported by the sequence logos. The phylogenetic analysis revealed the presence of magnoliophyte- and bryophyte-specific subgroups. We analyzed the public microarray data for expression of oleosin, caleosin and steroleosin in Arabidopsis and rice during the vegetative and reproductive stages, or under abiotic stresses. Our results indicated that genes encoding oleosin, caleosin and steroleosin proteins were expressed predominantly in plant seeds. This work may facilitate better understanding of the members of lipid-body-membrane proteins in diverse organisms and their gene expression in model plants Arabidopsis and rice.

Genome-wide comprehensive analysis of human helicases.

Helicases are motor proteins that catalyze the unwinding of duplex nucleic acids in an ATP-dependent manner. They are involved in almost all the nucleic acid transactions. In the present study, we report a comprehensive analysis of helicase gene family in human and its comparison with homologs in model organisms. The human genome encodes for 95 non-redundant helicase proteins, of which 64 are RNA helicases and 31 are DNA helicases. 57 RNA helicases are validated based on annotations and occurrence of conserved helicase signature motifs. These include 14 DExH and 37 DExD subfamily members, six other members such as U5.snRNP, ATR-X, Suv3, FANCJ, and two of superkiller viralicidic activity 2-like helicases. 31 DNA helicases are also identified, which include RecQ, MCM and RuvB-like helicases. Finding a set of helicases in human and almost similar sequences in model organisms suggests that the "core" members of helicase gene family are highly conserved throughout evolution. The present study gives an overview of members of RNA and DNA helicases encoded by the human genome along with their conserved motifs, phylogeny and homologs in model organisms. The study on comparing these homologs will spread light on the organization and complexity of helicase gene family in model organisms. The comprehensive analysis of human helicases presented in this study will further provide an invaluable resource for elaborate biological research on these helicases.

Genome-wide analysis of lipoxygenase gene family in Arabidopsis and rice.

The enzymes called lipoxygenases (LOXs) can dioxygenate unsaturated fatty acids, which leads to lipoperoxidation of biological membranes. This process causes synthesis of signaling molecules and also leads to changes in cellular metabolism. LOXs are known to be involved in apoptotic (programmed cell death) pathway, and biotic and abiotic stress responses in plants. Here, the members of LOX gene family in Arabidopsis and rice are identified. The Arabidopsis and rice genomes encode 6 and 14 LOX proteins, respectively, and interestingly, with more LOX genes in rice. The rice LOXs are validated based on protein alignment studies. This is the first report wherein LOXs are identified in rice which may allow better understanding the initiation, progression and effects of apoptosis, and responses to bitoic and abiotic stresses and signaling cascades in plants.

Oxysterol binding proteins (OSBPs) and their encoding genes in Arabidopsis and rice.

Cell wall deposition, biosynthesis of steroid hormones, and maintenance of membrane composition and integrity, are some of the crucial functions of sterols in plants. Followed by their synthesis in the endoplasmic reticulum, the sterols accumulate in the plasma membrane. The concept of sterol trafficking in plant cell is not well understood. The oxysterol binding proteins are implicated in sterol transport in non-plant systems. In the study, the oxysterol binding proteins in Arabidopsis and rice are described and classified. The Arabidopsis genome encodes 12 oxysterol binding proteins-related proteins (ORPs) as compared to 6 oxysterol binding proteins (OSBPs/ORPs) in rice. The protein alignment studies reveal that amino acid sequences for oxysterol binding proteins are relatively well conserved in Arabidopsis and rice. The rice OSBPs are classified based on their phylogenetic relationship with Arabidopsis ORPs. The sequence LOGO built on LOC_Os03g16690 indicated presence of fingerprint region of amino acids "EQVSHHPP" for Arabidopsis and rice OSBPs/ORPs. The organization of pleckstrin homology domain is identified in several OSBPs/ORPs in Arabidopsis and rice. The Arabidopsis oligonucleotide array data is explored to understand the expression patterns of ORPs under 17 different experimental conditions. The analysis showed the expression of ORPs in Arabidopsis is necessarily under the control of biotic stress, chemical, elicitor, hormone, light intensity, abiotic stress, and temperature conditions. The linear mean signal values for Arabidopsis ORPs revealed their relative expression patterns in different developmental stages. The genes for ORP3C and ORP3B are highly expressed in all developmental stages that were analyzed. The present study thus indicates crucial functional role of the individual members of this gene family in different environmental stress conditions.

Subcellular localization of proteins of Oryza sativa L. in the model tobacco and tomato plants.

The cellular localization and molecular interactions are indicative of functions of a protein. The development of a simple and efficient method for subcellular localization of a protein is indispensable to elucidate gene function in plants. In this study, we assessed the feasibility of Agrobacterium-mediated transformation (agroinfiltration) of tobacco and tomato leaf tissue to follow intracellular targeting of proteins from rice fused to green fluorescent protein (GFP). For this, a simple in planta assay for subcellular localization of rice proteins in the heterologous host systems of tobacco and tomato leaf via transient transformation was developed. We have tested the applicability of this method by expressing GFP fusions of the putative antiphagocytic protein 1 (APP1) (OsAPP, LOC_Os03g56930) and ZOS3-18 - C2H2 zinc-finger protein (OsZF1, LOC_Os03g55540) from Oryza sativa L. subsp. japonica in tobacco and tomato leaf tissues. Our results demonstrate the suitability of GFP as a reporter in gene expression studies in tomato cv MicroTom. The use of GFP-fused proteins from rice for subcellular targeting in the heterologous hosts of tobacco and tomato plant systems has been confirmed.

Architectures of the unique domains associated with the DEAD-box helicase motif.

Helicases are motor proteins of biological system, which catalyze the opening of energetically stable duplex nucleic acids in an ATP-dependent manner and thereby are involved in almost all aspects of nucleic acid metabolism including cell cycle progression. They contain several conserved domains including the DEAD-box and also several unique domains associated with these. The Pfam database (http://pfam.janelia.org/) is a large collection of protein families, each represented by multiple sequence alignments and hidden Markov models (HMMs). A diverse range of proteins are found in nature, and the functional specificity to each protein, to a greater extent, is imparted by its domain architecture. To this extent, a DEAD-box ATP-dependent RNA helicase (LOC_Os01g36890; Genomic sequence length: 6284 nucleotides; CDS length: 1,299 nucleotides; Protein length: 432 amino acids) was studied. The protein sequence was imported for domain search on Pfam. This particular Pfam entry after covering a large proportion of sequences in the underlying database has generated a more comprehensive coverage across a wide range of phyla of the known domains that are associated with the typical DEAD-box helicase motif. A total of 362 domain architectures were recollected from the Pfam database for the Family: DEAD (PF00270). We have therefore systematically analyzed the domains closely associated with DEAD-motif, which occur in a variety of proteins and can provide insights into their function.

microRNA access to the target helicases from rice.

Major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs are single-stranded RNA molecules of around 22 nucleotides in length. Most miRNAs show imperfect homology with their targets. The biogenesis mechanisms of miRNAs are different for plant and animals. Silencing of genes by miRNAs may serve as an appropriate tool to speed-up analyses of gene functions in a post-genome era. We have identified in silico a set of miRNAs that control helicase gene expression by regulating its mRNA stability and translation in rice. Our analysis revealed that several rice helicases have distinct miRNA specificities. Such analyses will be a prerequisite to refining our understanding of target selection and regulation of helicase gene expression by miRNAs in rice. Further, we discuss recent findings on miRNA gene family and its gene structure, criteria for miRNA annotation and on miRNA biogenesis that involve transcription, processing and maturation of miRNAs.

Mulberry improvements via plastid transformation and tissue culture engineering.

The in vitro tissue culture and micropropagation studies for Morus spp., a pivotal sericulture plant, are well established. The rapid and reproducible in vitro response to plant growth regulator treatments has emerged as an essential complement of transformation studies for this plant species. A major area of study is the use of protoplast culture and fusion techniques where advantages to mulberry improvement can be applied. The advancements in genetic transformation of mulberry are reviewed, and a section on strategy for transforming plastids (chloroplasts) of mulberry is included. A role for mulberry in "molecular farming" is envisioned. The conclusions and future prospects for improvement of this economically important tree species are proposed.

Conserved thioredoxin fold is present in Pisum sativum L. sieve element occlusion-1 protein.

Homology-based three-dimensional model for Pisum sativum sieve element occlusion 1 (Ps.SEO1) (forisomes) protein was constructed. A stretch of amino acids (residues 320 to 456) which is well conserved in all known members of forisomes proteins was used to model the 3D structure of Ps.SEO1. The structural prediction was done using Protein Homology/analogY Recognition Engine (PHYRE) web server. Based on studies of local sequence alignment, the thioredoxin-fold containing protein [Structural Classification of Proteins (SCOP) code d1o73a_], a member of the glutathione peroxidase family was selected as a template for modeling the spatial structure of Ps.SEO1. Selection was based on comparison of primary sequence, higher match quality and alignment accuracy. Motif 1 (EVF) is conserved in Ps.SEO1, Vicia faba (Vf.For1) and Medicago truncatula (Mt.SEO3); motif 2 (KKED) is well conserved across all forisomes proteins and motif 3 (IGYIGNP) is conserved in Ps.SEO1 and Vf.For1.

Genome-wide analysis of helicase gene family from rice and Arabidopsis: a comparison with yeast and human.

Helicases are motor proteins which can catalyze the unwinding of stable RNA or DNA duplex utilizing mainly ATP as source of energy. In this study we have identified complete sets of helicases from rice and Arabidopsis. The helicase gene family in rice and Arabidopsis contains 115 and 113 genes respectively. These helicases were validated based on their annotations and supported with organization of conserved helicase signature motifs. We have also identified homologs of 64 rice RNA and DNA helicases in Arabidopsis, yeast and human. We explored Arabidopsis oligonucleotide array data to gain functional insights into the transcriptome of helicase family members under ten different stress conditions. Our results revealed that expression of helicase genes is profoundly regulated under various stress conditions. The helicases identified in this study lay a foundation for the in depth characterization of each helicase type.

Forisomes as calcium-energized protein complex: a historical perspective.

Forisomes are spindle-shaped (about 1-3 µm wide and 10-30 µm long) contractile moving protein bodies found exclusively in phloem sieve tubes of Fabaceae (legume) plants. They are composed of ATP-independent, mechanically active proteins with several subunits called "forisomettes" and play important role in plant defence against injury. They expand and contract reversibly independent of ATP in response to changes of concentration of Ca2+ ions and pH or electric field. Although forisomes have been discovered decades ago, but only recently they received broader attention due to their ability to convert chemical into mechanical energy. Forisomes have possible applications as biomimetic smart materials (e.g. valves in microdevices), or smart composite materials for nanotechnological applications. Forisome-based smart materials can be used to synthesize self-powered monitoring and diagnostic systems for health monitoring. Here we discuss precise overview of forisomes from a historical viewpoint. We also enlist the scientific events leading to the identification and characterization of forisomes and their potential applications. The advancements in forisomes research have marked the evolution of new trends in the field of applied 'biomimetics' (means: to 'mimic life').

Genome-wide analysis of the family of light-harvesting chlorophyll a/b-binding proteins in Arabidopsis and rice.

Light-harvesting antenna system possesses an inherent property of photoprotection. The single-helix proteins found in cyanobacteria play role in photoprotection and/or pigment metabolism. The photoprotective functions are also manifested by the two- and four-helix proteins. The photoprotection mechanism evolved earlier to the mechanism of light-harvesting of the antenna complex. Here, the light-harvesting complex genes of photosystems I and II from Arabidopsis are enlisted, and almost similar set of genes are identified in rice. Also, the three-helix early light-inducible proteins (ELIPs), two-helix stress-enhanced proteins (SEPs), and one-helix high light-inducible proteins [one-helix proteins (OHPs)] are identified in rice. Interestingly, two independent genomic loci encoding PsbS protein are also identified with implications on additional mode of non-photochemical quenching (NPQ) mechanism in rice. A few additional LHC-related genes are also identified in rice (LOC_Os09g12540, LOC_Os02g03330). This is the first report of identification of light-harvesting complex genes and light-inducible genes in rice.

Genome-wide analysis of thioredoxin fold superfamily peroxiredoxins in Arabidopsis and rice.

A broad range of peroxides generated in subcellular compartments, including chloroplasts, are detoxified with peroxidases which are called as peroxiredoxins (Prx). The Prx are ubiquitously distributed in all organisms including bacteria, fungi, animals, and also in cyanobacteria and plants. Recently, the Prx have emerged as new molecules in antioxidant defense in plants. Here, the members which belong to Prx gene family in Arabidopsis and rice are been identified. Overall, the Prx members constitute a small family with 10 and 11 genes in Arabidopsis and rice respectively. The prx genes of rice are assigned to their functional groups based on homology search against Arabidopsis protein database. Deciphering the Prx functions in rice will add novel information to the mechanism of antioxidant defense in plants. Further, the Prx also forms the part of redox signaling cascade. Here, the Prx family has been described for rice.

Forisomes: calcium-powered protein complexes with potential as 'smart' biomaterials.

Sieve tubes in legumes contain forisomes, which are spindle-like bodies that are composed of ATP-independent, mechanically active proteins. Upon injury, forisomes occlude sieve tubes by dispersion and thus, help to prevent loss of nutrient-rich transport sap. Forisome enlargement by dispersion is brought about by Ca2+-induced conformational changes that confer radial expansion and longitudinal contraction. Forisomes recontract upon Ca2+ removal. In vitro, forisomes reversibly disperse and contract in the presence or absence of Ca2+, respectively, and at distinct pHs. Recently, forisomes have received renewed attention because of their unique capacity to convert chemical into mechanical energy independent of high-energy organic compounds. Forisome-based 'smart' materials can be used to produce self-powered monitoring and diagnostic systems. Here, we focus on physiological, chemical and physical aspects of forisomes and discuss their potential as biomimetic devices.

Impact of PsbTc on forward and back electron flow, assembly, and phosphorylation patterns of photosystem II in tobacco.

Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn(4)Ca cluster to the plastoquinone pool. It is a dimeric supramolecular complex comprising more than 30 subunits per monomer, of which 16 are bitopic or peripheral, low-molecular-weight components. Directed inactivation of the plastid gene encoding the low-molecular-weight peptide PsbTc in tobacco (Nicotiana tabacum) does not prevent photoautotrophic growth. Mutant plants appear normal green, and levels of PSII proteins are not affected. Yet, PSII-dependent electron transport, stability of PSII dimers, and assembly of PSII light-harvesting complexes (LHCII) are significantly impaired. PSII light sensitivity is moderately increased and recovery from photoinhibition is delayed, leading to faster D1 degradation in DeltapsbTc under high light. Thermoluminescence emission measurements revealed alterations of midpoint potentials of primary/secondary electron-accepting plastoquinone of PSII interaction. Only traces of CP43 and no D1/D2 proteins are phosphorylated, presumably due to structural changes of PSII in DeltapsbTc. In striking contrast to the wild type, LHCII in the mutant is phosphorylated in darkness, consistent with its association with PSI, indicating an increased pool of reduced plastoquinone in the dark. Finally, our data suggest that the secondary electron-accepting plastoquinone of PSII site, the properties of which are altered in DeltapsbTc, is required for oxidation of reduced plastoquinone in darkness in an oxygen-dependent manner. These data present novel aspects of plastoquinone redox regulation, chlororespiration, and redox control of LHCII phosphorylation.

Deletion of PsbM in tobacco alters the QB site properties and the electron flow within photosystem II.

Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.

PsbI affects the stability, function, and phosphorylation patterns of photosystem II assemblies in tobacco.

Photosystem II (PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q(A) binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redox-regulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in delta psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently.

Construction, database integration, and application of an Oenothera EST library.

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.