The Predicted Functional Compartmentation of Rice Terpenoid Metabolism by Trans-Prenyltransferase Structural Analysis, Expression and Localization.

Most terpenoids are derived from the basic terpene skeletons of geranyl pyrophosphate (GPP, C10), farnesyl-PP (FPP, C15) and geranylgeranyl-PP (GGPP, C20). The trans-prenyltransferases (PTs) mediate the sequential head-to-tail condensation of an isopentenyl-PP (C5) with allylic substrates. The in silico structural comparative analyses of rice trans-PTs with 136 plant trans-PT genes allowed twelve rice PTs to be identified as GGPS_LSU (OsGGPS1), homomeric G(G)PS (OsGPS) and GGPS_SSU-II (OsGRP) in Group I; two solanesyl-PP synthase (OsSPS2 and 3) and two polyprenyl-PP synthases (OsSPS1 and 4) in Group II; and five FPSs (OsFPS1, 2, 3, 4 and 5) in Group III. Additionally, several residues in "three floors" for the chain length and several essential domains for enzymatic activities specifically varied in rice, potentiating evolutionarily rice-specific biochemical functions of twelve trans-PTs. Moreover, expression profiling and localization patterns revealed their functional compartmentation in rice. Taken together, we propose the predicted topology-based working model of rice PTs with corresponding terpene metabolites: GPP/GGPPs mainly in plastoglobuli, SPPs in stroma, PPPs in cytosol, mitochondria and chloroplast and FPPs in cytosol. Our findings could be suitably applied to metabolic engineering for producing functional terpene metabolites in rice systems.

Physical-Chemical Crosslinked Electrospun Colocasia esculenta Tuber Protein-Chitosan-Poly(Ethylene Oxide) Nanofibers with Antibacterial Activity and Cytocompatibility.

BACKGROUND: Electrospun nanofibers based on Colocasia esculenta tuber (CET) protein are considered as a promising material for wound dressing applications. However, the use of these nanofibers in aqueous conditions has poor stability. The present study was performed to obtain insights into the crosslinked electrospun CET's protein-chitosan (CS)-poly(ethylene oxide) (PEO) nanofibers and to evaluate their potential for wound dressing applications. METHODS: The electrospun nanofibers were crosslinked with glutaraldehyde (GA) vapor and heat treatment (HT) to enhance their physicochemical stability. The crosslinked nanofibers were characterized by protein profiles, morphology structures, thermal behavior, mechanical properties, and degradation behavior. Furthermore, the antibacterial properties and cytocompatibility were analyzed by antibacterial assessment and cell proliferation. RESULTS: The protein profiles of the electrospun CET's protein-CS-PEO nanofibers before and after HT crosslinking contained one major bioactive protein with a molecular weight of 14.4 kDa. Scanning electron microscopy images of the crosslinked nanofibers indicated preservation of the structure after immersion in phosphate buffered saline. The crosslinked nanofibers resulted in higher ultimate tensile strength and lower ultimate strain compared to the non-crosslinked nanofibers. GA vapor crosslinking showed higher water stability compared to HT crosslinking. The in vitro antibacterial activity of the crosslinked nanofibers showed a stronger bacteriostatic effect on Staphylococcus aureus than on Escherichia coli. Human skin fibroblast cell proliferation on crosslinked GA vapor and HT nanofibers with 1% (w/v) CS and 2% (w/v) CET's protein demonstrated the highest among all the other crosslinked nanofibers after seven days of cell culture. Cell proliferation and cell morphology results revealed that introducing higher CET's protein concentration on crosslinked nanofibers could increase cell proliferation of the crosslinked nanofibers. CONCLUSION: These results are promising for the potential use of the crosslinked electrospun CET's protein-CS-PEO nanofibers as bioactive wound dressing materials.

Unusual features of the c-ring of F1FO ATP synthases.

Membrane integral ATP synthases produce adenosine triphosphate, the universal "energy currency" of most organisms. However, important details of proton driven energy conversion are still unknown. We present the first high-resolution structure (2.3 Å) of the in meso crystallized c-ring of 14 subunits from spinach chloroplasts. The structure reveals molecular mechanisms of intersubunit contacts in the c14-ring, and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane. Similar densities were found in all known high-resolution structures of c-rings of F1FO ATP synthases from archaea and bacteria to eukaryotes. The densities might originate from isoprenoid quinones (such as coenzyme Q in mitochondria and plastoquinone in chloroplasts) that is consistent with differential UV-Vis spectroscopy of the c-ring samples, unusually large distance between polar/apolar interfaces inside the c-ring and universality among different species. Although additional experiments are required to verify this hypothesis, coenzyme Q and its analogues known as electron carriers of bioenergetic chains may be universal cofactors of ATP synthases, stabilizing c-ring and prevent ion leakage through it.

Structure of a Kunitz-type potato cathepsin D inhibitor.

Potato cathepsin D inhibitor (PDI) is a glycoprotein of 188 amino acids which can inhibit both the aspartic protease cathepsin D and the serine protease trypsin. Here we report the first X-ray structure of PDI at a resolution of 2.1 Å showing that PDI adopts a ß-trefoil fold, which is typical of the Kunitz-family protease inhibitors, with the inhibitory loops protruding from the core. Possible reactive-site loops including one involving a unique disulphide and another involving a protruding 310 helix are identified and docking studies indicate the mode of action of this unusual bi-functional inhibitor.

Interfacial and emulsifying properties of soybean peptides with different degrees of hydrolysis.

In this study, the effects of the degree of hydrolysis on the interfacial and emulsifying properties of soybean peptides were evaluated based on surface and interfacial tension, dynamic light scattering (DLS), and freeze-fracture transmission electron microscopy (FF-TEM) analyses. Of the five evaluated soybean peptides (SP95, SP87, SP75, SP49, and SP23), those with higher degrees of hydrolysis (SP95 and SP87) did not exhibit noticeable surface-active properties in water, whereas those with relatively low degrees of hydrolysis (SP75, SP49, and SP23) exhibited remarkable surface tension-lowering activity. The latter set (SP75, SP49, and SP23) also formed giant associates with average sizes ranging from 64.5 nm to 82.6 nm above their critical association concentration (CAC). Moreover, SP23 with the lowest degree of hydrolysis exhibited excellent emulsifying activity for soybean oil, and FF-TEM analysis demonstrated that the emulsions were stabilized by a lamella-like multilayer peptide structure on the oil droplets that prevented coagulation. The peptide with the lowest degree of hydrolysis (SP23) was effective not only for soybean oil emulsification, but also for the emulsification of liquid paraffin and silicon oil that are generally difficult to emulsify.

Inactivation, aggregation, secondary and tertiary structural changes of germin-like protein in Satsuma mandarine with high polyphenol oxidase activity induced by ultrasonic processing.

The inhibition of Polyphenol oxidase (PPO) in plants has been widely researched for their important roles in browning reaction. A newly found germin-like protein (GLP) with high PPO activity in Satsuma mandarine was inactivated by low-frequency high-intensity ultrasonic (20 kHz) processing. The effects of ultrasound on PPO activity and structure of GLP were investigated using dynamic light scattering (DLS) analysis, transmission electron microscopy (TEM), circular dichroism (CD) spectral measurement and fluorescence spectral measurement. The lowest PPO activity achieved was 27.4% following ultrasonication for 30 min at 400 W. DLS analysis showed ultrasound caused both aggregation and dissociation of GLP particles. TEM images also demonstrated protein aggregation phenomena. CD spectra exhibited a certain number of loss in α-helix structure content. Fluorescence spectra showed remarkable increase in fluorescence intensity with tiny blue-shift following ultrasonication. In conclusion, ultrasound applied in this study induced structural changes of GLP and eventually inactivated PPO activity.

Role of γ-kafirin in the formation and organization of kafirin microstructures.

The possible importance of the cysteine-rich γ-prolamin in kafirin and zein functionality has been neglected. The role of γ-kafirin in organized microstructures was investigated in microparticles. Residual kafirin (total kafirin minus γ-kafirin) "microparticles" were non-discrete (amorphous mass of material), as viewed by electron microscopy and atomic force microscopy. Adding 15% γ-kafirin to residual kafirin resulted in the formation of a mixture of non-discrete material and nanosize discrete spherical structures. Adding 30% γ-kafirin to the residual kafirin resulted in discrete spherical nanosize particles. Fourier transform infrared spectroscopy indicated that γ-kafirin had a mixture of random-coil and ß-sheet conformations, in contrast to total kafirin, which is mainly α-helical conformation. γ-Kafirin also had a very high glass transition temperature (Tg) (≈270 °C). The conformation and high Tg of γ-kafirin probably confer structural stability to kafirin microstructures. Because of its ability to form disulfide cross-links, γ-kafirin appears to be essential to form and stabilize organized microstructures.

ATP-driven remodeling of the linker domain in the dynein motor.

Dynein ATPases are the largest known cytoskeletal motors and perform critical functions in cells: carrying cargo along microtubules in the cytoplasm and powering flagellar beating. Dyneins are members of the AAA+ superfamily of ring-shaped enzymes, but how they harness this architecture to produce movement is poorly understood. Here, we have used cryo-EM to determine 3D maps of native flagellar dynein-c and a cytoplasmic dynein motor domain in different nucleotide states. The structures show key sites of conformational change within the AAA+ ring and a large rearrangement of the "linker" domain, involving a hinge near its middle. Analysis of a mutant in which the linker "undocks" from the ring indicates that linker remodeling requires energy that is supplied by interactions with the AAA+ modules. Fitting the dynein-c structures into flagellar tomograms suggests how this mechanism could drive sliding between microtubules, and also has implications for cytoplasmic cargo transport.

Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis.

In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.

Distinct roles of 1alpha and 1beta heavy chains of the inner arm dynein I1 of Chlamydomonas flagella.

The Chlamydomonas I1 dynein is a two-headed inner dynein arm important for the regulation of flagellar bending. Here we took advantage of mutant strains lacking either the 1α or 1ß motor domain to distinguish the functional role of each motor domain. Single- particle electronic microscopic analysis confirmed that both the I1α and I1ß complexes are single headed with similar ringlike, motor domain structures. Despite similarity in structure, however, the I1ß complex has severalfold higher ATPase activity and microtubule gliding motility compared to the I1α complex. Moreover, in vivo measurement of microtubule sliding in axonemes revealed that the loss of the 1ß motor results in a more severe impairment in motility and failure in regulation of microtubule sliding by the I1 dynein phosphoregulatory mechanism. The data indicate that each I1 motor domain is distinct in function: The I1ß motor domain is an effective motor required for wild-type microtubule sliding, whereas the I1α motor domain may be responsible for local restraint of microtubule sliding.

Three outer arm dynein heavy chains of Chlamydomonas reinhardtii operate in a coordinated fashion both in vitro and in vivo.

Outer arm dynein (OAD) in cilia and flagella contains two to three nonidentical heavy chains (HCs) that possess motor activity. In Chlamydomonas, flagellar OAD contains three HCs, alpha-, beta-, and gamma-HCs, each appearing to have a distinct role. To determine the precise molecular mechanism of their function, cross-sectional electron micrographs of wild-type and single HC-disruption mutants were compared and statistically analyzed. While the alpha-HC mutant displayed an OAD of lower density, which was attributed to a lack of alpha-HC, the OAD of beta- and gamma-HC mutants not only lacked the corresponding HC, but was also significantly affected in its structure, particularly with respect to the localization of alpha-HC. The lack of beta-HC induced mislocalization of alpha-HC, while a disruption of the gamma-HC gene resulted in the synchronized movement of alpha-HC and beta-HC in the manners for stacking. Interestingly, using cryo-electron microscopy, purified OADs were typically observed consisting of two stacked heads and an independent single head, which presumably corresponded to gamma-HC. This conformation is different from previous reports in which the three HCs displayed a stacked form in flagella observed by cryo-electron tomography and a bouquet structure on mica in deep-etch replica images. These results suggest that gamma-HC supports the tight stacking arrangement of inter or intra alpha-/beta-HC to facilitate the proper functioning of OAD.

GTP is required for the microtubule catastrophe-inducing activity of MAP200, a tobacco homolog of XMAP215.

Widely conserved among eukaryotes, the microtubule-associated protein 215 (MAP215) family enhances microtubule dynamic instability. The family member studied most extensively, Xenopus laevis XMAP215, has been reported to enhance both assembly and disassembly parameters, although the mechanism whereby one protein can exert these apparently contradictory effects has not been clarified. Here, we analyze the activity of a plant MAP215 homolog, tobacco (Nicotiana tabacum) MAP200 on microtubule behavior in vitro. We show that, like XMAP215, MAP200 promotes both assembly and disassembly parameters, including microtubule growth rate and catastrophe frequency. When MAP200 is added to tubulin and taxol, strikingly long-coiled structures form. When GDP partially replaces GTP, the increase of catastrophe frequency by MAP200 is strongly diminished, even though this replacement stimulates catastrophe in the absence of MAP200. This implies that MAP200 induces catastrophes by a specific, GTP-requiring pathway. We hypothesize that, in the presence of MAP200, a catastrophe-prone microtubule lattice forms occasionally when elongated but nonadjacent protofilaments make lateral contacts.

Crystallisation, structure and function of plant light-harvesting Complex II.

The chlorophyll a/b light-harvesting complex of photosystem II (LHC-II) collects most of the solar energy in the biosphere. LHC-II is the prototype of a highly conserved family of membrane proteins that fuels plant photosynthesis in the conversion of excitation energy into biologically useful chemical energy. In addition, LHC-II plays an important role in the organisation of the thylakoid membrane, the structure of the photosynthetic apparatus, the regulation of energy flow between the two photosystems, and in the controlled dissipation of excess excitation energy under light stress. Our current understanding of the sophisticated mechanisms behind each of these processes has profited greatly from the progress made over the past two decades in determining the structure of the complex. This review presents the developments and breakthroughs that ultimately lead to the high-resolution structure of LHC-II. Based on an alignment of the remarkably well engineered and highly conserved LHC polypeptide, we propose several key features of the LHC-II structure that are likely to be present in all members of the LHC family. Finally, some recently proposed mechanisms of energy-dependent non-photochemical quenching (NPQ) are examined from a structural perspective.

Characterization of Phaseolus vulgaris L. landraces cultivated in central Italy.

Eight Phaseolus vulgaris L. landraces cultivated on farm in marginal areas of Central Italy (Lazio region) were investigated in order to evaluate chemical composition of storage proteins and secondary metabolites fractions. The total protein content showed some differences among landraces; the maximum value was next to 30 g for 100 g of dry weight. The seed storage proteins were screened by polyacrylamide gel electrophoresis (SDS/PAGE): seven landraces exhibited phaseolin patterns type S, one landrace showed a phaseolin pattern type T. A morphological analysis of cotyledon parenchyma performed by scanning electron microscopy (SEM) revealed differences in size of starch granules. Moreover the polyphenolic composition was investigated using HPLC-APCI; from the methanol extracts a flavonoid, kaempferol, and a coumarin, 5,7-dimethoxycoumarin, were identified. To our knowledge, this is the first time that 5,7-dimethoxycoumarin has been reported in P. vulgaris seeds.

Filament formation and robust strand exchange activities of the rice DMC1A and DMC1B proteins.

The DMC1 protein, a meiosis-specific DNA recombinase, catalyzes strand exchange between homologous chromosomes. In rice, two Dmc1 genes, Dmc1A and Dmc1B, have been reported. Although the Oryza sativa DMC1A protein has been partially characterized, however the biochemical properties of the DMC1B protein have not been defined. In the present study, we expressed the Oryza sativa DMC1A and DMC1B proteins in bacteria and purified them. The purified DMC1A and DMC1B proteins formed helical filaments along single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and promoted robust strand exchange between ssDNA and dsDNA over five thousand base pairs in the presence of RPA, as a co-factor. The DMC1A and DMC1B proteins also promoted strand exchange in the absence of RPA with long DNA substrates containing several thousand base pairs. In contrast, the human DMC1 protein strictly required RPA to promote strand exchange with these long DNA substrates. The strand-exchange activity of the Oryza sativa DMC1A protein was much higher than that of the DMC1B protein. Consistently, the DNA-binding activity of the DMC1A protein was higher than that of the DMC1B protein. These biochemical differences between the DMC1A and DMC1B proteins may provide important insight into their functional differences during meiosis in rice.

Engineered resistance against proteinases.

Exogenous proteinase inhibitors are valuable and economically interesting protective biotechnological tools. We examined whether small proteinase inhibitors when fused to a selected target protein can protect the target from proteolytic degradation without simultaneously affecting the function and activity of the target domain. Two proteinase inhibitors were studied: a Kazal-type silk proteinase inhibitor (SPI2) from Galleria mellonella, and the Cucurbita maxima trypsin inhibitor I (CMTI I). Both inhibitors target serine proteinases, are small proteins with a compact structure stabilized by a network of disulfide bridges, and are expressed as free polypeptides in their natural surroundings. Four constructs were prepared: the gene for either of the inhibitors was ligated to the 5' end of the DNA encoding one or the other of two selected target proteins, the coat protein (CP) of Potato potyvirus Y or the Escherichia coli beta-glucuronidase (GUS). CMTI I fused to the target proteins strongly hampered their functions. Moreover, the inhibitory activity of CMTI I was retained only when it was fused to the CP. In contrast, when fused to SPI2, specific features and functions of both target proteins were retained and the inhibitory activity of SPI2 was fully preserved. Measuring proteolysis in the presence or absence of either inhibitor, we demonstrated that proteinase inhibitors can protect target proteins used either free or as a fusion domain. Interestingly, their inhibitory efficiency was superior to that of a commercial inhibitor of serine proteinases, AEBSF.

A thermostable cysteine protease precursor from a tropical plant contains an unusual C-terminal propeptide: cDNA cloning, sequence comparison and molecular modeling studies.

We report here the cloning and characterization of the entire cDNA of a papain-like cysteine protease from a tropical flowering plant. The 1098-bp ORF of the cDNA codify a protease precursor having a signal peptide of 19 amino acids, a cathepsin-L like N-terminal proregion of 114 amino acids, a mature enzyme part of 208 amino acids and a C-terminal proregion of 24 amino acids. The derived amino acid sequence of the mature part tallies with the thermostable cysteine protease Ervatamin-C--as was aimed at. The C-terminal proregion of the protease has altogether a different sequence pattern not observed in other members of the family and it contains a negatively charged helical zone. The three-dimensional model of the precursor, based on the homology modeling and X-ray structure, shows that the extended peptide stretch region of the N-terminal propeptide, covering the interdomain cleft, contains protruding side chains of positively charged residues. This study also indicates that the negatively charged zone of C-terminal propeptide may interact with the positively charged zone of the N-terminal propeptide in a cooperative manner in the maturation process of this enzyme.

Localization of vacuolar transport receptors and cargo proteins in the Golgi apparatus of developing Arabidopsis embryos.

Using immunogold electron microscopy, we have investigated the relative distribution of two types of vacuolar sorting receptors (VSR) and two different types of lumenal cargo proteins, which are potential ligands for these receptors in the secretory pathway of developing Arabidopsis embryos. Interestingly, both cargo proteins are deposited in the protein storage vacuole, which is the only vacuole present during the bent-cotyledon stage of embryo development. Cruciferin and aleurain do not share the same pattern of distribution in the Golgi apparatus. Cruciferin is mainly detected in the cis and medial cisternae, especially at the rims where storage proteins aggregate into dense vesicles (DVs). Aleurain is found throughout the Golgi stack, particularly in the trans cisternae and trans Golgi network where clathrin-coated vesicles (CCVs) are formed. Nevertheless, aleurain was detected in both DV and CCV. VSR-At1, a VSR that recognizes N-terminal vacuolar sorting determinants (VSDs) of the NPIR type, localizes mainly to the trans Golgi and is hardly detectable in DV. Receptor homology-transmembrane-RING H2 domain (RMR), a VSR that recognizes C-terminal VSDs, has a distribution that is very similar to that of cruciferin and is found in DV. Our results do not support a role for VSR-At1 in storage protein sorting, instead RMR proteins because of their distribution similar to that of cruciferin in the Golgi apparatus and their presence in DV are more likely candidates. Aleurain, which has an NPIR motif and seems to be primarily sorted via VSR-At1 into CCV, also possesses putative hydrophobic sorting determinants at its C-terminus that could allow the additional incorporation of this protein into DV.

Localization of arabinogalactan proteins in anther, pollen, and pollen tube of Nicotiana tabacum L.

Western blot analysis indicated the presence of two epitopes recognized by the anti-arabinogalactan protein antibodies JIM13 and LM2 and the absence of the JIM4 epitope in mature tobacco anthers. Immunoenzyme localization of arabinogalactan proteins (AGPs) with JIM13 showed that AGPs accumulate mainly at the early stages of anther development. AGP content and distribution were also investigated at the ultrastructural level in pollen tubes grown in vivo and in vitro. Abundant AGPs were present in the transmitting tissue of styles, and the AGP content of the extracellular matrix changed during pollen tube growth. In pollen tubes, immunogold particles were mainly distributed in the cell wall and cytoplasm, especially around the peripheral region of the generative-cell wall. beta-D-Glucosyl Yariv reagent, which specifically binds to AGPs, caused slow growth of pollen tubes and reduced immunogold labeling of AGPs with JIM13 in vitro. These data suggest that AGPs participate in male gametogenesis and pollen tube growth and may be important surface molecules in generative and sperm cells.

Structural properties of caleosin: a MS and CD study.

We have investigated the covalent and secondary solution structure of caleosin, a 27-kDa protein also called ATS1 or AtClo1 (At4g26740) found within Arabidopsis thaliana seed lipid bodies. The native protein was partly phosphorylated at S225. Purified bacterially expressed caleosin (recClo) was not phosphorylated; cysteine residues C221 and C230 were connected by a disulfide bridge. In solution it exists as a mixture of predominant monomers and covalent dimers. We have used recClo as a model for the study of AtClo1 secondary structure. recClo is folded in aqueous solution (16% alpha-helix, 29% beta-sheet), its secondary structure being dramatically influenced by the polarity of media, as deduced from CD spectra measured in the presence of increasing concentrations of various aliphatic alcohols.