PLANT PROTEINS

Transcription factor IIICZ (TFIIICZ), together with other transcription factors (TFIIIB and TFIIICl), is required for the in vitro transcription of tRNA and adenovirus VA genes by RNA polymerase 111. Previous studies have shown that TFIIICZ is a high molecular weight (-500,000) protein which binds with high affinity to the B-box promoter element of tRNA-type genes. A polypeptide of M , -250,000 is in close association with DNA in the specific complex between TFIIICZ and the B-box promoter element. Here we describe the purification of TFIIICZ by a factor of -25,000 from nuclear extracts of HeLa cells by ionic exchange, affinity, and hydrophobic chromatography and sedimentation velocity centrifugation. The most purified fractions contain polypeptides of approximately 230 kDa (corresponding to the polypeptide which can be cross-linked to VA1 DNA), 110,100,80, and 60 kDa which co-sediment with TFIIICZ B-box specific binding and in vitro transcriptional activities.

Phenylpropenes such as chavicol, t-anol, eugenol, and isoeugenol are produced by plants as defense compounds against animals and microorganisms and as floral attractants of pollinators. Moreover, humans have used phenylpropenes since antiquity for food preservation and flavoring and as medicinal agents. Previous research suggested that the phenylpropenes are synthesized in plants from substituted phenylpropenols, although the identity of the enzymes and the nature of the reaction mechanism involved in this transformation have remained obscure. We show here that glandular trichomes of sweet basil (Ocimum basilicum), which synthesize and accumulate phenylpropenes, possess an enzyme that can use coniferyl acetate and NADPH to form eugenol. Petunia (Petunia hybrida cv. Mitchell) flowers, which emit large amounts of isoeugenol, possess an enzyme homologous to the basil eugenol-forming enzyme that also uses coniferyl acetate and NADPH as substrates but catalyzes the formation of isoeugenol. The basil and petunia phenylpropene-forming enzymes belong to a structural family of NADPH-dependent reductases that also includes pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase.

The cloning of the jellyfish gfp (green fluorescent protein) gene and its alteration for expression in subcellular locations in transformed plant cells have resulted in new views of intracellular organization and dynamics. Fusions of GFP with entire proteins of known or unknown function have shown where the proteins are located and whether the proteins move from one compartment to another. GFP and variants with different spectral properties have been deliberately targeted to separate compartments to determine their size, shape, mobility, and dynamic changes during development or environmental response. Fluorescence Resonance Energy Transfer (FRET) between GFP variants can discern proteinu protein interactions. GFP has been used as a sensor to detect changes or differences in calcium, pH, voltage, metal, and enzyme activity. Photobleaching and photoactivation of GFP as well as fluorescence correlation spectroscopy can measure rates of diffusion and movement of GFP within or between compartments. This review covers past applications of these methods as well as promising developments in GFP imaging for understanding the functional organization of plant cells.

The R gene family determines the timing, distribution and amount of anthocyanin pigmentation in maize. This family comprises a set of regulatory genes, consisting of a cluster of several elements at the R locus, on chromosome 10, the Lc and Sn gene lying about two units Rdistal and 8 o n chromosome 2. Each gene determines a tissue-specific pigmentation of different parts of the seed and plant. The proposed duplicated function of R, Sn, Lc and B loci is reflected in cDNA sequence similarity. In this paper an extensive analysis of the predicted proteins of the R, Sn, Lc and B genes together with a search for putative sites of post-translational modification is reported. A comparison with the PROSITE database discloses several Nglycosylation and phosphorylation sites, as well as the basic Helix-Loop-Helix (HLH) domain of transcriptional activators. Sn, Lc, and R S show a high conservation of these sites, while B is more divergent. Analysis of the 5' leader of mRNA sequences discloses the presence of five ATG triplets with two upstream open reading frames (uORFs) of 38 and 15 amino acids and a loop structure indicating a possible mechanism of control at the translational level. It is conceivable that possible mechanisms acting at the translational and post-translational level could modulate the expression and the activation of these transcription factors. Northern analysis of various tissues of different R alleles highlights a strict correlation between pigment accumulation in different tissues and the expression of the regulatory and structural genes suggesting that the pattern of pigmentation relies on a mechanism of differential expression of the members of the Rfamily. Analysis of the Sn promoter discloses the presence of several sequences resembling binding sites of known transcription factors (as GAGA and GT) that might be responsible for the spatial and light-induced expression

The biased amino acid composition and aperiodic (random coil) configuration of Group 1 late embryogenesisabundant (LEA) proteins imply that these proteins are capable of binding large amounts of water. While Group 1 LEAs have been predicted to contribute to osmotic stress protection in both embryonic and vegetative tissues, biochemical support has been lacking. We have used Saccharomyces cerevisiae as a model system to test the putative osmoprotective function of a wheat Group 1 LEA protein, Em. We demonstrate that expression of Em protein in yeast cells is not deleterious to growth in media of normal osmolarity and attenuates the growth inhibition normally observed in media of high osmolarity. Enhanced growth is observed in the presence of a variety of osmotically active compounds indicating that Em protein is capable of mitigating the detrimental effect of low water potential in a relatively non-specific manner. These results are the first biochemical demonstration of an osmoprotective function for a Group 1 LEA and suggest that the yeast expression system will be useful in dissecting the mechanism of protection through structure-function studies.

Tyrosinases from various organisms are compared with respect to enzymatic structure. primary, secondary and tertiary structure, domain structure, Cu binding sites, maturation mechanism and activation mechanism. On the basis of these comparisons, and by using hemocyanin structure as a template, a structure model for the active site of tyrosinases is proposed.

Photosystem II reaction centers evolve 02 in the dark when H 202 is added as a substrate. Although some of this activity can be attributed to catalase, as much as 75% of the activity was not affected by the addition of 1 mM KCN. Several lines of evidence demonstrate that this KCN-insensitive 02 evolution from H 202 in the dark is catalyzed by the cycling of S states in the oxygen-evolving complex including: (1) inactivation of H2Oz-mediated 02 evolution by Ca/EDTA washing; (2) susceptibility of the activity to inhibition by amines like ammonia and Tris; (3) inhibition by CCCP which is known to accelerate the rate of deactivation of the S 2 state and; (4) a direct dependence of the rate of 02 evolution on the presence of calcium and (5) chloride.

Pine nuts are a part of traditional cooking in many parts of the world and have seen a significant increase in availability/use in the United States over the past 10 years. The U.S. Food and Drug Administration (US FDA) field offices received 411 complaints from U.S. consumers over the past three years regarding taste disturbances following the consumption of pine nuts. Using analysis of fatty acids by gas chromatography with flame ionization detection, previous reports have implicated nuts from Pinus armandii (Armand Pine) as the causative species for similar taste disturbances. This method was found to provide insufficient species resolution to link FDA consumer complaint samples to a single species of pine, particularly when samples contained species mixtures of pine nuts. Here we describe a DNA based method for differentiating pine nut samples using the ycf1 chloroplast gene. Although the exact cause of pine nut associated dysgeusia is still not known, we found that 15 of 15 samples from consumer complaints contained at least some Pinus armandii, confirming the apparent association of this species with taste disturbances.

The compact (dwarf) plant architecture is an important trait in cucumber (Cucumis sativus L.) breeding that has the potential to be used in once-over mechanical harvest of cucumber production. Compact growth habit is controlled by a simply inherited recessive gene cp. With 150 F 2:3 families derived from two inbred cucumber lines, PI 308915 (compact vining) and PI 249561 (regular vining), we conducted genome-wide molecular mapping with microsatellite (simple sequence repeat, SSR) markers. A framework genetic map was constructed consisting of 187 SSR loci in seven linkage groups (chromosomes) covering 527.5 cM. Linkage analysis placed cp at the distal half of the long arm of cucumber Chromosome 4. Molecular markers cosegregating with the cp locus were identified through whole genome scaffold-based chromosome walking. Fine genetic mapping with 1,269 F 2 plants delimited the cp locus to a 220 kb genomic DNA region. Annotation and function prediction of genes in this region identified a homolog of the cytokinin oxidase (CKX) gene, which may be a potential candidate of compact gene. Alignment of the CKX gene homologs from both parental lines revealed a 3-bp deletion in the first exon of PI 308915, which can serve as a marker for marker-assisted selection of the compact phenotype. This work also provides a solid foundation for map-based cloning of the compact gene and understanding the molecular mechanisms of the dwarfing in cucumber.

PM28A is a major intrinsic protein of the spinach leaf plasma membrane and the major phosphoprotein. Phosphorylation of PM28A is dependent in vivo on the apoplastic water potential and in vitro on submicromolar concentrations of Ca 2 ؉ . Here, we demonstrate that PM28A is an aquaporin and that its water channel activity is regulated by phosphorylation. Wild-type and mutant forms of PM28A, in which putative phosphorylation sites had been knocked out, were expressed in Xenopus oocytes, and the resulting increase in osmotic water permeability was measured in the presence or absence of an inhibitor of protein kinases (K252a) or of an inhibitor of protein phosphatases (okadaic acid). The results indicate that the water channel activity of PM28A is regulated by phosphorylation of two serine residues, Ser-115 in the first cytoplasmic loop and Ser-274 in the C-terminal region. Labeling of spinach leaves with 32 P-orthophosphate and subsequent sequencing of PM28A-derived peptides demonstrated that Ser-274 is phosphorylated in vivo, whereas phosphorylation of Ser-115, a residue conserved among all plant plasma membrane aquaporins, could not be demonstrated. This identifies Ser-274 of PM28A as the amino acid residue being phosphorylated in vivo in response to increasing apoplastic water potential and dephosphorylated in response to decreasing water potential. Taken together, our results suggest an active role for PM28A in maintaining cellular water balance.

Members of the abscisic acid-responsive element binding protein (AREB)/abscisic acid-responsive element binding factor (ABF) subfamily of basic leucine zipper (bZIP) transcription factors have been implicated in abscisic acid (ABA) and abiotic stress responses in plants. Here we describe two members identified in cultivated tomato (Solanum lycopersicum), named SlAREB1 and SlAREB2. Expression of SlAREB1 and SlAREB2 is induced by drought and salinity in both leaves and root tissues, although that of SlAREB1 was more affected. In stress assays, SlAREB1-overexpressing transgenic tomato plants showed increased tolerance to salt and water stress compared to wild-type and SlAREB1-down-regulating transgenic plants, as assessed by physiological parameters such as relative water content (RWC), chlorophyll fluorescence and damage by lipoperoxidation. In order to identify SlAREB1 target genes responsible for the enhanced tolerance, microarray and cDNA-amplified fragment length polymorphism (AFLP) analyses were performed. Genes encoding oxidative stress-related proteins, lipid transfer proteins (LTPs), transcription regulators and late embryogenesis abundant proteins were found among the up-regulated genes in SlAREB1-overexpressing lines, especially in aerial tissue. Notably, several genes encoding defence proteins associated with responses to biotic stress (e.g. pathogenesis-related proteins, protease inhibitors, and catabolic enzymes) were also up-regulated by SlAREB1 overexpression, suggesting that this bZIP transcription factor is involved in ABA signals that participate in abiotic stress and possibly in response to pathogens.

Plant disease resistance genes operate at the earliest steps of pathogen perception. The Arabidopsis RPP5 gene specifying resistance to the downy mildew pathogen Peronospora parasitica was positionally cloned. It encodes a protein that possesses a putative nucleotide binding site and leucine-rich repeats, and its product exhibits striking structural similarity to the plant resistance gene products N and L6. Like N and L6, the RPP5 N-terminal domain resembles the cytoplasmic domains of the Drosophila Toll and mammalian interleukin-1 transmembrane receptors. In contrast to N and L6, which produce predicted truncated products by alternative splicing, RPP5 appears to express only a single transcript corresponding to the full-length protein. However, a truncated form structurally similar to those of N and L6 is encoded by one or more other members of the RPP5 gene family that are tightly clustered on chromosome 4. The organization of repeated units within the leucine-rich repeats encoded by the wild-type RPP5 gene and an RPP5 mutant allele provides molecular evidence for the heightened capacity of this domain to evolve nove1 configurations and potentially new disease resistance specificities.

The leaf economics spectrum (LES) represents a suite of intercorrelated leaf traits concerning construction costs per unit leaf area, nutrient concentrations, and rates of carbon fixation and tissue turnover. Although broad trade-offs among leaf structural and physiological traits have been demonstrated, we still do not have a comprehensive view of the fundamental constraints underlying the LES trade-offs. Here, we investigated physiological and structural mechanisms underpinning the LES by analysing a novel data compilation incorporating rarely considered traits such as the dry mass fraction in cell walls, nitrogen allocation, mesophyll CO2 diffusion and associated anatomical traits for hundreds of species covering major growth forms. The analysis demonstrates that cell wall constituents are major components of leaf dry mass (18-70%), especially in leaves with high leaf mass per unit area (LMA) and long lifespan. A greater fraction of leaf mass in cell walls is typically associated...

Quantitative information on growing organs is required to better understand morphogenesis in both plants and animals. however, detailed analyses of growth patterns at cellular resolution have remained elusive. We developed an approach, multiangle image acquisition, three-dimensional reconstruction and cell segmentation-automated lineage tracking (mArs-Alt), in which we imaged whole organs from multiple angles, computationally merged and segmented these images to provide accurate cell identification in three dimensions and automatically tracked cell lineages through multiple rounds of cell division during development. using these methods, we quantitatively analyzed Arabidopsis thaliana flower development at cell resolution, which revealed differential growth patterns of key regions during early stages of floral morphogenesis. lastly, using rice roots, we demonstrated that this approach is both generic and scalable.

The effects on performance, digestibility, N utilization and plasma amino acid concentrations of dietary chickpea (Cicer arietinum, var. Kabuli) seed meal, globulin proteins or buffer-insoluble residue (starch / non-starch polysaccharides (NSP)/ lignin) were studied in growing rats. Chickpea meal, defatted soybean meal, chickpea globulins and lactalbumin were each incorporated into diets as the sole source of dietary protein (100 g/kg).

Self-incompatibility (SI) is a mechanism for preventing self-fertilization in flowering plants. In Brassica, it is controlled by a single multi-allelic locus, S, and it is believed that two highly polymorphic genes in the S locus, SLG and SRK, play central roles in self-recognition in stigmas. SRK is a putative receptor protein kinase, whose extracellular domain exhibits high similarity to SLG. We analyzed two pairs of lines showing cross-incompatibility (S 2 and S 2-b ; S 13 and S 13-b ). In S 2 and S 2-b , SRKs were more highly conserved than SLGs. This was also the case with S 13 and S 13-b . This suggests that the SRKs of different lines must be conserved for the lines to have the same self-recognition specificity. In particular, SLG 2-b showed only 88.5% identity to SLG 2 , which is comparable to that between the SLGs of different S haplotypes, while SRK 2-b showed 97.3% identity to SRK 2 in the S domain. These findings suggest that the SLGs in these S haplotypes are not important for self-recognition in SI. Corresponding author: Makoto Kusaba, Institute of Radiation Breed-SRK (Conner et al. 1997; Stahl et al. 1998) reduced ing, National Institute of Agrobiological Resources, P.O. Box 3, Ohmiya-machi, Naka-gun, Ibaraki-ken, 319-2293, Japan. expression of SLG and SRK and caused conversion to

Autofluorescent protein tags represent one of the major and, perhaps, most powerful tools in modern cell biology for visualization of various cellular processes in vivo. In addition, advances in confocal microscopy and the development of autofluorescent proteins with different excitation and emission spectra allowed their simultaneous use for detection of multiple events in the same cell. Nevertheless, while autofluorescent tags are widely used in plant research, the need for a versatile and comprehensive set of vectors specifically designed for fluorescent tagging and transient and stable expression of multiple proteins in plant cells from a single plasmid has not been met by either the industrial or the academic communities. Here, we describe a new modular satellite (SAT) vector system that supports N-and C-terminal fusions to five different autofluorescent tags, EGFP, EYFP, Citrine-YFP, ECFP, and DsRed2. These vectors carry an expanded multiple cloning site that allows easy exchange of the target genes between different autofluorescence tags, and expression of the tagged proteins is controlled by constitutive promoters, which can be easily replaced with virtually any other promoter of interest. In addition, a series of SAT vectors has been adapted for high throughput Gateway recombination cloning. Furthermore, individual expression cassettes can be assembled into Agrobacterium binary plasmids, allowing efficient transient and stable expression of multiple autofluorescently tagged proteins from a single vector following its biolistic delivery or Agrobacteriummediated genetic transformation.

Cereals are an important part of diets for hypercholesterolemic patients. However, some of these patients are allergic to these natural products. The purpose of the current study was to compare oatmeal with equal in nutritional values two allergy-free amaranth meals to determine whether this pseudocereal can be a substitute for allergic to cereals individuals. The total phenols of the samples were determined with the Folin-Chocalteu reagent, anthocyanins, and flavonoids spectrophotometrically. The antioxidant activities were estimated with nitric oxide scavenging radical (NO) and by ␤-carotene bleaching (␤-carotene). It was found that the contents of different protein fractions, antioxidant compounds, and the antioxidant activities of oatmeal were significantly higher than those of the two amaranth samples. The results of kinetic reactions showed that samples differed in their capacities to quench these radicals, and oats have shown more antioxidant activity than amaranth. High correlation was observed between antioxidant activities and phenols (R 2 ϭ 0.99). In the in vivo part of the investigation, 60 male Wistar rats were divided into five diet groups of 12 animals each; these groups were designated as Control, Chol, Chol/Oat, Chol/AmarI, and Chol/AmarII. The rats of the Control group were fed basal diet (BD) only. To the BD of the four other groups were added the following: 1% of cholesterol (Chol), 10% of oat meal and 1% of cholesterol (Chol/Oat), 10% of amaranth I meal, and 1% of cholesterol (Chol/AmarI) and 10% of amaranth II meal and 1% of cholesterol (Chol/AmarII). After 32 days of different feeding, diets supplemented with oat meal and, to lesser degree, with amaranth I and amaranth II hindered the rise in the plasma lipids: a) TC: 3.14 vs. 4.57 mmol/L, Ϫ 31.3%; 3.31 vs. 4.57 mmol/L Ϫ 27.6%; and 3.40 vs. 4.57, Ϫ 25.6%, respectively b) LDL-C: 1.69 vs. 3.31 mmol/L, Ϫ 49.9%; 2.05 vs. 3.31 mmol/L, Ϫ 38.1%; and 2.16 vs. 3.31 mmol/L, Ϫ 34.8%, respectively; c) TG: 0.73 vs. 0.88 mmol/L, Ϫ 17.1%; 0.75 vs. 0.88 mmol/L, Ϫ 14.8%; and 0.79 vs. 0.88 mmol/L, Ϫ10.2%, respectively. The HDL-PH was increased as follows: 0.79 vs. 0.63 mmol/L, Ϫ25.3%; 0.75 vs. 0.63 mmol/L, Ϫ23.0%; and 0.71 vs. 0.63 mmol/L, Ϫ12

Anthocyanin concentration is a primary determinant of plant colour. Fruit anthocyanin biosynthesis is controlled by a distinct clade of R2R3 MYB transcription factors. In apple, three recent papers describe the discovery of MYB genes activating skin, flesh and foliage anthocyanic colour. These findings lead the way to new approaches in the breeding and biotechnological development of fruit with new colour patterns.

Pungency in Capsicum fruits is due to the accumulation of the alkaloid capsaicin and its analogs. The biosynthesis of capsaicin is restricted to the genus Capsicum and results from the acylation of an aromatic moiety, vanillylamine, by a branched-chain fatty acid. Many of the enzymes involved in capsaicin biosynthesis are not well characterized and the regulation of the pathway is not fully understood. Based on the current pathway model, candidate genes were identified in public databases and the literature, and genetically mapped. A published EST co-localized with the Pun1 locus which is required for the presence of capsaicinoids. This gene, AT3, has been isolated and its nucleotide sequence has been determined in an array of genotypes within the genus. AT3 showed significant similarity to acyltransferases in the BAHD superfamily. The recessive allele at this locus contains a deletion spanning the promoter and first exon of the predicted coding region in every non-pungent accession tested. Transcript and protein expression of AT3 was tissue-specific and developmentally regulated. Virus-induced gene silencing of AT3 resulted in a decrease in the accumulation of capsaicinoids, a phenotype consistent with pun1. In conclusion, gene mapping, allele sequence data, expression profile and silencing analysis collectively indicate that the Pun1 locus in pepper encodes a putative acyltransferase, and the pun1 allele, used in pepper breeding for nearly 50 000 years, results from a large deletion at this locus.

Fibrillins are nuclear-encoded, plastid proteins associated with chromoplast fibrils and chloroplast plastoglobules, thylakoids, photosynthetic antenna complexes, and stroma. There are 12 sub-families of fibrillins. However, only three of these sub-families have been characterized genetically or functionally. We review evidence indicating that fibrillins are involved in plastoglobule structural development, chromoplast pigment accumulation, hormonal responses, protection of the photosynthetic apparatus from photodamage, and plant resistance to a range of biotic and abiotic stresses. The area of fibrillin research has substantial growth potential and will contribute to better understanding of mechanisms of plant stress tolerance and plastid structure and function.

A variety of labdane-related diterpenoids, including phytocassanes, oryzalexins and momilactones, were identified as phytoalexins in rice (Oryza sativa L.). Momilactone B was also isolated as an allelochemical exuded from rice roots. The biosynthetic genes of these phytoalexins have been identified, including six labdane-related diterpene cyclase genes such as OsCPS2, OsCPS4, OsKSL4, OsKSL7, OsKSL8 and OsKSL10. Here we identified an OsCPS4 knockdown mutant, cps4-tos, by screening Tos17 mutant lines using polymerase chain reaction. OsCPS4 encodes a syn-copalyl diphosphate synthase responsible for momilactones and oryzalexin S biosynthesis. Because Tos17 was inserted into the third intron of OsCPS4, the mature OsCPS4 mRNA was detected in the cps4-tos mutant as well as the wild type. Nevertheless, mature OsCPS4 transcript levels in the cps4-tos mutant were about one sixth those in the wild type. The cps4-tos mutant was more susceptible to rice blast fungus than the wild type, possibly due to lower levels of momilactones and oryzalexin S in the mutant. Moreover, co-cultivation experiments suggested that the allelopathic effect of cps4-tos against some kinds of lowland weeds was significantly lower than that of the wild type, probably because of lower momilactone content exuded from cps4-tos roots. A reverse-genetic strategy using the cps4-tos mutant showed the possible roles of momilactones not only as phytoalexins but also as allelopathic substances.

Background: Root-knot nematodes are sedentary endoparasites that can infect more than 3000 plant species. Root-knot nematodes cause an estimated $100 billion annual loss worldwide. For successful establishment of the root-knot nematode in its host plant, it causes dramatic morphological and physiological changes in plant cells. The expression of some plant genes is altered by the nematode as it establishes its feeding site. Results: We examined the expression of soybean (Glycine max) genes in galls formed in roots by the root-knot nematode, Meloidogyne incognita, 12 days and 10 weeks after infection to understand the effects of infection of roots by M. incognita. Gene expression was monitored using the Affymetrix Soybean GeneChip containing 37,500 G. max probe sets. Gene expression patterns were integrated with biochemical pathways from the Kyoto Encyclopedia of Genes and Genomes using PAICE software. Genes encoding enzymes involved in carbohydrate and cell wall metabolism, cell cycle control and plant defense were altered. Conclusions: A number of different soybean genes were identified that were differentially expressed which provided insights into the interaction between M. incognita and soybean and into the formation and maintenance of giant cells. Some of these genes may be candidates for broadening plants resistance to root-knot nematode through over-expression or silencing and require further examination.

Vegetable proteins are an integral part of infant weaning diets in Latin America. Protein quality in plant-based products, however, is constrained by amino acid composition and intrinsically present antinutritional factors. The goal of this study was to improve bean protein quality by utilizing fermentation and germination processing. The objectives were to determine if protein quality, as measured by Food and Agricultural Organization (FAO) approved True Protein Digestibility (TPD) and Protein Digestibility-Corrected Amino Acid Scores (PDCAAS), of formulated bean-based weaning products could be improved upon fermentation and germination and if protein quality could be further improved when processed beans were combined with cooked rice. Results showed that the highest TPD and PDCAAS values were obtained for cooked germinated beans combined with rice. The TPD values for products ranged from 80 to 91%, and the PDCAAS values were 0.38-0.51. There was no significant increase (P < 0.05) of either TPD or PDCAAS values upon fermentation. Germination increased TPD of cooked bean products; this increase was not, however, accompanied by an increase in PDCAAS. When combined with rice, the PDCAAS values for all bean products improved significantly, thus supporting the concept of cereal-legume complementation. In conclusion, this study showed the range of PDCAAS in processed black bean and bean-rice infant weaning food products. The potential for incorporation of these products into the diets of weaning age Latin American children would, however, be confirmed only after validation with growth or metabolic balance studies in human infants.

Our study aimed at investigating the influence of elevated atmospheric CO 2 concentration on the salinity tolerance of the cash crop halophyte Aster tripolium L., thereby focussing on protein expression and enzyme activities. The plants were grown in hydroponics using a nutrient solution with or without addition of NaCl (75% seawater salinity), under ambient (380 ppm) and elevated (520 ppm) CO 2 . Under ambient CO 2 concentration enhanced expressions and activities of the antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and glutathione-S-transferase in the salt-treatments were recorded as a reaction to oxidative stress. Elevated CO 2 led to significantly higher enzyme expressions and activities in the salt-treatments, so that reactive oxygen species could be detoxified more effectively. Furthermore, the expression of a protective heat shock protein (class 20) increased under salinity and was even further enhanced under elevated CO 2 concentration. Additional energy had to be provided for the mechanisms mentioned above, which was indicated by the increased expression of a b ATPase subunit and higher v-, p-and f-ATPase activities under salinity. The higher ATPase expression and activities also enable a more efficient ion transport and compartmentation for the maintenance of ion homeostasis. We conclude that elevated CO 2 concentration is able to improve the survival of A. tripolium under salinity because more energy is provided for the synthesis and enhanced activity of enzymes and proteins which enable a more efficient ROS detoxification and ion compartmentation/transport.

The genome of the bladderwort Utricularia gibba provides an unparalleled opportunity to uncover the adaptive landscape of an aquatic carnivorous plant with unique phenotypic features such as absence of roots, development of water-filled suction bladders, and a highly ramified branching pattern. Despite its tiny size, the U. gibba genome accommodates approximately as many genes as other plant genomes. To examine the relationship between the compactness of its genome and gene turnover, we compared the U. gibba genome with that of four other eudicot species, defining a total of 17,324 gene families (orthogroups). These families were further classified as either 1) lineage-specific expanded/contracted or 2) stable in size. The U. gibba-expanded families are generically related to three main phenotypic features: 1) trap physiology, 2) key plant morphogenetic/developmental pathways, and 3) response to environmental stimuli, including adaptations to life in aquatic environments. Further scans for signatures of protein functional specialization permitted identification of seven candidate genes with amino acid changes putatively fixed by positive Darwinian selection in the U. gibba lineage. The Arabidopsis orthologs of these genes (AXR, UMAMIT41, IGS, TAR2, SOL1, DEG9, and DEG10) are involved in diverse plant biological functions potentially relevant for U. gibba phenotypic diversification, including 1) auxin metabolism and signal transduction, 2) flowering induction and floral meristem transition, 3) root development, and 4) peptidases. Taken together, our results suggest numerous candidate genes and gene families as interesting targets for further experimental confirmation of their functional and adaptive roles in the U. gibba's unique lifestyle and highly specialized body plan. by guest on February 15, 2015 http://gbe.oxfordjournals.org/ Downloaded from NOTE.-B, M and C indicate biological process, molecular function and cellular component GO classes, respectively. Sample counts and genome counts indicate the total numbers of genes annotated with particular GO terms among expanded families (of which there are 1,754 genes total) versus the entire U. gibba genome (which contains a total of 28,494 genes). P-values resulting from Fisher exact tests are shown (in italic, those significant after Benjamini-Hochberg correction). Selected GO terms generically related to 1) trap physiology, 2) plant morphogenetic/developmental pathways, and 3) response to environmental stimuli and adaptations to life in aquatic environments are colored orange, green, and blue, respectively. Adaptive Molecular Evolution in U. gibba GBE Genome Biol. Evol. 7(2):444-456.

There has been an increasing interest in the development of systematic methods for the synthesis of purification steps for biotechnological products, which are often the most difficult and costly stages in a biochemical process. Chromatographic processes are extensively used in the purification of multicomponent biotechnological systems. One of the main challenges in the synthesis of purification processes is the appropriate selection and sequencing of chromatographic steps that are capable of producing the desired product at an acceptable cost and quality. This paper describes mathematical models and solution strategies based on mixed integer linear programming (MILP) for the synthesis of multistep purification processes. First, an optimization model is proposed that uses physicochemical data on a protein mixture, which contains the desired product, to select a sequence of operations with the minimum number of steps from a set of candidate chromatographic techniques that must achieve a specified purity level. Since several sequences that have the minimum number of steps may satisfy the purity level, it is possible to obtain the one that maximizes final purity. Then, a second model that may use the total number of steps obtained in the first model generates a solution with the maximum purity of the product. Whenever the sequence does not affect the final purity or more generally does not impact the objective function, alternative models that are of smaller size are developed for the optimal selection of steps. The models are tested in several examples, containing up to 13 contaminants and a set of 22 candidate high-resolution steps, generating sequences of six operations, and are compared to the current synthesis approaches.

We have studied the distribution of the protein synthesis inhibitory activity in the tissues of Saponaria officinalis L. (Caryophyllaceae). Seven major saporins, ribosome-inactivating proteins, were purified to apparent homogeneity from leaves, roots and seeds using a new procedure of RIPs isolation including ion-exchange and hydrophobic chromatography. They all catalysed the depurination of rat liver ribosomes, which generate the Endo's diagnostic rRNA fragment upon treatment with acid aniline, thus indicating that m 4324 from the 28S rRNA has been released (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). The molecular mass of saporins by SDS-PAGE ranged between 30.2 and 31.6 kDa and by gel-filtration between 27.5 and 30.1 kDa. Amino acid composition and amino-terminal amino acid sequence indicate that all saporins may be considered isoforms. Only two saporins present in roots were glycosylated (SO-R1 and SO-R3). All saporins are very active on cell-free translation systems derived from rabbit reticulocyte lysates, rat liver, Triticum aestivum L., Cucumis sativus L. and Vicia sativa L. However, they are poor inhibitors of an Escherichia coli translation system. They inhibit protein synthesis in HeLa, BeWo and NB 100 cells, HeLa cells being the most resistant. The enzymatic activity of at least one saporin isoform was dependent on magnesium concentration in the standard rat liver cell-free system.

Ca 2+ -calmodulin (Ca 2+ -CaM) is a critical molecule that mediates cellular functions by interacting with various metabolic and signaling pathways. However, the protein expression patterns and accompanying serial cytological responses in Ca 2+ -CaM signaling deficiency remain enigmatic. Here, we provide a global analysis of the cytological responses and significant alterations in protein expression profiles after trifluoperazine treatment in Picea meyeri, which abrogates Ca 2+ -CaM signaling. Ninety-three differentially displayed proteins were identified by comparative proteomics at different development stages and were assigned to different functional categories closely related to tip growth machinery. The inhibition of Ca 2+ -CaM signaling rapidly induced an increase in extracellular Ca 2+ influx, resulting in dramatically increased cytosolic Ca 2+ concentrations and ultrastructural abnormalities in organelles as the primary responses. Secondary and tertiary alterations included actin filament depolymerization, disrupted patterns of endocytosis and exocytosis, and cell wall remodeling, ultimately resulting in perturbed pollen tube extension. In parallel with these cytological events, time-course experiments revealed that most differentially expressed proteins showed time-dependent quantitative changes (i.e. some signaling proteins and proteins involved in organelle functions and energy production changed first, followed by alterations in proteins related to cytoskeletal organization, secretory pathways, and polysaccharide synthesis). Taken together, Ca 2+ -CaM dysfunction induced serial cytological responses and temporal changes in protein expression profiles, indicating the pivotal role of Ca 2+ -CaM in the regulation of tip growth machinery.

Pathogen recognition by the plant immune system is governed by structurally related, polymorphic products of disease resistance (R) genes. RAR1 and/or SGT1b mediate the function of many R proteins. RAR1 controls preactivation R protein accumulation by an unknown mechanism. We demonstrate that Arabidopsis SGT1b has two distinct, genetically separable functions in the plant immune system: SGT1b antagonizes RAR1 to negatively regulate R protein accumulation before infection, and SGT1b has a RAR1-independent function that regulates programmed cell death during infection. The balanced activities of RAR1 and SGT1, in concert with cytosolic HSP90, modulate preactivation R protein accumulation and signaling competence.

The conformation of puroindoline-a and -b, two basic lipid-binding proteins isolated from wheat seedlings, has been studied for the first time by infrared and Raman spectroscopy. The infrared results show that puroindoline-a and -b have similar secondary structure composed of approximately 30% R-helices, 30% -sheets, and 40% unordered structure at pH 7. The conformation of both puroindolines is significantly pH-dependent. The reduction of the disulfide bridges leads to a decrease of the solubility of puroindolines in water and to an increase of the -sheet content by about 15% at the expense of the R-helix content. Raman spectroscopy confirms the structure similarity between the two puroindolines with little differences in the side chains' environment. All the disulfide bridges are in a gauche-gauchegauche conformation, and the unique tyrosine residue present in both puroindolines is hydrogen-bonded to water. Raman spectra have been recorded in both H 2 O and D 2 O media, thus providing additional information concerning the accessibility of certain residues to water. We have also observed that puroindoline-a tends to form some aggregates under acidic and high ionic strength conditions. Nearultraviolet circular dichroism measurements suggest that the tryptophan-rich domain is involved in this aggregate formation. Finally, on the basis of a combined infrared and sequence conformational analysis, we propose a secondary structure assignment for both puroindolines. The results show that puroindolines exhibit a similar folding pattern with plant nonspecific lipid-transfer protein and some amylase-protease inhibitors. These proteins could form a homogeneous structural family of plant proteins involved in the defense against pathogens that are probably derived from a common "helicoidal" protein ancestor. † This research was supported in part by research grants from the Natural Sciences and Engineering Research Council of Canada (grant to M.P.), the Fonds pour la formation de chercheurs et l'aide à la recherche of the Province of Quebec (grant to M.P.), and grants from Region pays de Loire and Ministère de l'enseignement et de la recherche

The radiate sunflower inflorescence is composed by zygomorphic ray flowers and actinomorphic disk flowers. Studies performed on mutants identify HaCYC2c, a CYCLOIDEA (CYC)-like gene, as one of the key players controlling flower symmetry in sunflower. turf and tub mutants are characterized by a shift from zygomorphic to actinomorphic ray flowers, caused by insertion of transposable elements (TEs) in HaCYC2c gene. In dbl or Chry mutants, an insertion upstream the coding region of HaCYC2c causes the ectopic expression of the gene and the shift from actinomorphic to zygomorphic disk flowers. We focused on Chry2 mutant: a 1034 bp insertion placed 558 bp before the start codon of HaCYC2c was identified. The insertion is a truncated version of a CACTA TE. Unexpectedly, phenotypic and genetic co-segregation analysis in F 2 and F 3 progenies derived from the crosses Chry2 3 turf and turf 3 Chry2 demonstrated that CACTA insertion is not always sufficient to alter the expression of HaCYC2c gene and generate Chry2 phenotype. F 3 plants homozygous for the CACTA insertion displayed either HaCYC2c transcription pattern identical to wild-type plants or a normal heterogamous inflorescence. Stated these results, we conclude that a much more complex regulatory system stays behind the Chry2 phenotype. genesis 52:315-327,

This paper reports the identification and functional expression of a gene that is involved in nitrate uptake in plants, a process essential for the assimilation of nitrate and the biological removal of nitrate from the soil solution. The CM1 gene of Arabidopsis, which when mutated confers resistance to the herbicide chlorate and a decrease in nitrate uptake, was isolated and found to encode a protein with 12 putative membrane-spanning segments. Injection of CM1 mRNA into Xenopus oocytes produces a nitrate-and pHdependent membrane depolarization, inward current, and nitrate uptake. These data show that the CM.7 gene encodes an electrogenic nitrate transporter. CM7 mRNA is found predominantly in roots and displays nitrate-and pH-dependent regulation.

Successful defense depends on the ability of the plant to recognize an attacking 'enemy' as early as possible. Early defense responses require enemy-initiated signaling cascades. Their activation ensures an induced response that is quantitative, timely and coordinated with other activities of the host cells. Damage-induced ion imbalances and modulations of channel activities are the first events occurring in the plasma membrane and result in rapid perturbations of the plasma membrane potential (V m ) involving variations of cytosolic Ca 2+ concentrations. Interacting downstream networks of kinases and phytohormones mediate the signal and result in concerted gene activation. Here we review and discuss early events occurring before herbivore attack-related gene expression that are responsible for cascades of events and signal transductions, eventually leading to indirect and direct plant responses.

The crystal structure of Hg(I1)-plastocyanin h v been determined and refined at a resolution of 1.9 A. The crystals were prepared by soaking crystals of Cu(I1)plastocyanin from poplar leaves (Populus nigra var. italica) in a solution of a mercuric salt. Replacement of the Cu(I1) atom in plastocyanin by Hg(I1) causes only minor changes in the geometry of the metal site, and there are few significant changes elsewhere in the molecule. It is concluded that, as in the case of the native protein, the geometry of the metal site is determined by the polypeptide. The weak metal-S(methionine) bond found in Cu(I1)-plastocyanin remains weak in Hg(I1)-plastocyanin. The "flip" of a proline side chain close to the metal site from a Cy-exo conformation in Cu(I1)-plastocyanin to a C7-endo conformation in Hg(I1)-plastocyanin suggests that this region of the molecule is particularly flexible.

Phylogenetic relationships of the five families of the order Commelinales remain an area of deep uncertainty in higher-level monocot systematics, despite intensive morphological and anatomical study. To test the monophyly of the Commelinales and the subclass Commelinidae, evaluate their relationships, and analyze evolutionary trends in their morphology, ecology, and biogeography, we conducted parsimony analyses on 95 rbcL sequences representing 17 taxa of Commelinales, 16 taxa of other Commelinidae, and 63 taxa from Arecidae, Liliidae, and Zingiberidae. Commelinales is polyphyletic and Commelinidae paraphyletic, with Eriocaulaceae and Xyridaceae sister to Poaceae and its relatives, Rapateaceae sister to Bromeliaceae and Mayacaceae, and Commelinaceae sister to Philydrales and allies. Thurnia is sister to Prionium at the base of Cyperaceae-Juncaceae; only 1 of Cronquist's multifamily commelinoid orders is diagnosed as monophyletic. We propose a revised Commelinidae, incorporating 4 revised superorders (Bromelianae, Commelinanae, Dasypogonanae, Arecanae) and 10 orders ((Poales, Eriocaulales, Cyperales, Typhales, Bromeliales), (Commelinales, Philydrales, Zingiberales), (Dasypogonales), (Arecales)). Morphological and anatomical characters used to define the original Commelinales and Commelinidae appear to be plesiomorphic or to reflect convergence or recurrent mutation; several characters supporting our revised classification are anatomical traits that seem relatively insulated from environmental selection pressures. The Commelinidae distal to the Arecales arose in South America, with amphiatlantic Bromeliaceae-Mayacaceae-Rapateaceae originating in the Guayana Shield. Ecological diversification involved the repeated invasion of shady, infertile, or arid microsites. The numbers of species in families of the revised Commelinidae are related partly to the extent of adaptive radiation in those families, but seem more strongly related to nonadaptive features promoting speciation, such as restricted seed dispersal (especially in forest interior groups with fleshy fruits), polyploidy, aneuploidy, and apomixis. Species diversity is unrelated to the rate/amount of rbcL sequence evolution. 1999 Academic Press

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant protein inhibitors of endo-polygalacturonases (PGs) that belong to the leucine-rich repeat (LRR) protein family. In bean, PGIP is encoded by a small gene family of four members among which Pvpgip2 encodes the most wide-spectrum and efficient inhibitor of fungal PGs. In order to evaluate the sequence polymorphism of Pvpgip2 and its functional significance, we have analyzed a number of wild and cultivated bean (P. vulgaris) accessions of Andean and Mesoamerican origin, and some genotypes from the related species P. coccineus, P. acutifolius, and P. lunatus. Our analyses indicate that the protein encoded by Pvpgip2 is highly conserved in the bean germplasm. The few detected polymorphic sites correspond to synonymous substitutions and only two wild genotypes contain a Pvpgip2 with a single non-synonymous replacement. Sequence comparison showed a slightly larger variation in the related bean species P. coccineus, P. ac...

Rubisco activase (Rca) is a chaperone-like protein of the AAA+ family, which uses mechano-chemical energy derived from ATP hydrolysis to release tightly bound inhibitors from the active site of the primary carbon fixing enzyme ribulose 1,5-bisphosphate oxygenase/carboxylase (Rubisco). Mechanistic and structural investigations of Rca have been hampered by its exceptional thermolability, high degree of size polydispersity and propensity towards subunit aggregation. In this work, we have characterized the thermal stability and self-association behavior of recombinant Rca preparations, and have developed ligand screening methods. Thermal denaturation profiles generated by circular dichroism indicate that creosote and tobacco short-form Rcas are the most stable proteins examined, with an estimated mid-point temperature of 45-47°C for protein denaturation. We demonstrate that ADP provides a higher degree of stabilization than ATP, that magnesium ions have a small stabilizing effect on ATP-bound, but a significant destabilizing effect on ADP-bound Rca, and that phosphate provides weak stabilization of the ADP-bound form of the protein. A dimeric species was identified by size-exclusion chromatography, suggesting that the two-subunit module may comprise the basic building block for larger assemblies. Evidence is provided that chromatographic procedures reflect non-equilibrium multimeric states. Dynamic light scattering experiments performed on nucleotide-bearing Rca support the notion that several larger, highly polydisperse assembly states coexist over a broad concentration range. No significant changes in aggregation are observed upon replacement of ADP with ATP. However, in the absence of nucleotides, the major protein population appears to consist of a monodisperse oligomer smaller than a hexamer.

The rising prevalence to food allergies in the past two decades, together with the fact that the only existing therapy is avoidance of allergen-containing food next to the implementation of anti-allergic drugs, urges the need for improved performance of current assays to detect potential allergens in food products. Therein, the focus has been on aptamer-based biosensors in recent years. In this paper we report for the first time the selection of aptamers against one of the most important peanut allergens, Ara h 1. Several Ara h1 DNA aptamers were selected after eight selection rounds using capillary electrophoresis (CE)-SELEX. The selected aptamers specifically recognized Ara h 1 and did not significantly bind with other proteins, including another peanut allergen Ara h 2. The dissociation constant of a best performing aptamer was in the nanomolar range as determined independently by three different approaches, which are surface plasmon resonance, fluorescence anisotropy, and capillary electrophoresis (353 7 82 nM, 419 763 nM, and 450 7 60 nM, respectively). Furthermore, the selected aptamer was used for bioassay development on a home-built fiber optic surface plasmon resonance (FO-SPR) biosensor platform for detecting Ara h 1 protein in both buffer and food matrix samples demonstrating its real potential for the development of novel, more accurate aptamer-based biosensors. In conclusion, the reported aptamer holds a great potential for the detection of Ara h 1 in both the medical field and the food sector due to its high affinity and specificity for the target protein.

Dioscorin, the storage protein of yam (Dioscorea batatas Decne) tuber (which is different from dioscorine found in tubers of Dioscorea hirsuta), was purified to homogeneity after DE-52 ion exchange column according to the methods of Hou et al. (J. Agric. Food Chem. 1999, 47, 2168-2172. A single band of 32 kDa dioscorin was obtained on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel with 2-mercaptoethanol treatment. This purified dioscorin was shown by spectrophotometric method to have scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in a pH-dependent manner. There is a positive correlation between scavenging effects against DPPH (8-46%) and amounts of 32 kDa dioscorin (5.97-47.80 nmol) added in Tris-HCl buffer (pH 7.9), which are comparable to those of glutathione at the same concentrations. Using electron paramagnetic resonance (EPR) spectrometry for DPPH radical detection, it was found that the intensities of the EPR signal were decreased by 28.6 and 57 nmol of 32 kDa dioscorin in Tris-HCl buffer (pH 7.9) more than in distilled water compared to controls. EPR spectrometry was also used for hydroxyl radical detection. It was found that 32 kDa dioscorin could capture hydroxyl radical, and the intensities of the EPR signal were significantly decreased dose-dependently by 1.79-14.32 nmol of 32 kDa dioscorin (r ) 0.975) compared to the control. It is suggested that 32 kDa dioscorin, the storage protein of yam tuber, may play a role as antioxidant in tubers and may be beneficial for health when people take it as a food additive or consume yam tubers.

New insights into the phenomenon of systemic acquired resistance have been gained in recent years, by the use of techniques in molecular genetics and biology that have replaced the largely descriptive approach of earlier work. The isolation of mutants in the signal transduction pathway from induction to expression of resistance as well as the use of transgenic plants over-expressing or suppressing the expression of putative candidate genes involved in systemic acquired resistance and its signalling have identified several steps in the establishment of plant resistance. In this review the latest developments implicating salicylic acid as a signal molecule in systemic resistance are discussed and contrasted with new signalling pathways which, seemingly, are based on alternative mechanisms.

. Three cDNA clones encoding timothy grass pollen profilin Phl p 11 were newly isolated. Comparison of the sequences of four cDNA clones, including a previously isolated clone, showed a low level of polymorphism. Isoelectrofocusing of highly purified timothy grass profilin indicated the existence of at least five isoforms. One recombinant profilin showed similar immunological properties to natural timothy grass profilin. Tertiary structure of Phleum pratense profilin was obtained by homology-based molecular modeling. q 1997 Elsevier Science B.V.