Gibberellin Biosynthesis in Maize. Metabolic Studies with GA(15), GA(24), GA(25), GA(7), and 2,3-Dehydro-GA(9)

[17-(14)C]-Labeled GA(15), GA(24), GA(25), GA(7), and 2,3-dehydro-GA(9) were separately injected into normal, dwarf-1 (d1), and dwarf-5 (d5) seedlings of maize (Zea mays L.). Purified radioactive metabolites from the plant tissues were identified by full-scan gas chromatography-mass spectrometry and Kovats retention index data. The metabolites from GA(15) were GA(44), GA(19), GA(20), GA(113), and GA(15)-15,16-ene (artifact?). GA(24) was metabolized to GA(19), GA(20), and GA(17). The metabolites from GA(25) were GA(17), GA(25) 16alpha,17-H(2)-17-OH, and HO-GA(25) (hydroxyl position not determined). GA(7) was metabolized to GA(30), GA(3), isoGA(3) (artifact?), and trace amounts of GA(7)-diene-diacid (artifact?). 2,3-Dehydro-GA(9) was metabolized to GA(5), GA(7) (trace amounts), 2,3-dehydro-GA(10) (artifact?), GA(31), and GA(62). Our results provide additional in vivo evidence of a metabolic grid in maize (i.e. pathway convergence). The grid connects members of a putative, non-early 3,13-hydroxylation branch pathway to the corresponding members of the previously documented early 13-hydroxylation branch pathway. The inability to detect the sequence GA(12) --> GA(15) --> GA(24) --> GA(9) indicates that the non-early 3,13-hydroxylation pathway probably plays a minor role in the origin of bioactive gibberellins in maize.     

A likelihood approach for comparing synonymous and nonsynonymous nucleotide substitution rates, with application to the chloroplast genome

A model of DNA sequence evolution applicable to coding regions is presented. This represents the first evolutionary model that accounts for dependencies among nucleotides within a codon. The model uses the codon, as opposed to the nucleotide, as the unit of evolution, and is parameterized in terms of synonymous and nonsynonymous nucleotide substitution rates. One of the model's advantages over those used in methods for estimating synonymous and nonsynonymous substitution rates is that it completely corrects for multiple hits at a codon, rather than taking a parsimony approach and considering only pathways of minimum change between homologous codons. Likelihood-ratio versions of the relative-rate test are constructed and applied to data from the complete chloroplast DNA sequences of Oryza sativa, Nicotiana tabacum, and Marchantia polymorpha. Results of these tests confirm previous findings that substitution rates in the chloroplast genome are subject to both lineage-specific and locus-specific effects. Additionally, the new tests suggest tha the rate heterogeneity is due primarily to differences in nonsynonymous substitution rates. Simulations help confirm previous suggestions that silent sites are saturated, leaving no evidence of heterogeneity in synonymous substitution rates.      

The Significance of Protein Maturation by Plastidic Type I Signal Peptidase 1 for Thylakoid Development in Arabidopsis Chloroplasts

Thylakoids are the chloroplast internal membrane systems that house light-harvesting and electron transport reactions. Despite the important functions and well-studied constituents of thylakoids, the molecular mechanism of their development remains largely elusive. A recent genetic study has demonstrated that plastidic type I signal peptidase 1 (Plsp1) is vital for proper thylakoid development in Arabidopsis (Arabidopsis thaliana) chloroplasts. Plsp1 was also shown to be necessary for processing of an envelope protein, Toc75, and a thylakoid lumenal protein, OE33; however, the relevance of the protein maturation in both of the two distinct subcompartments for proper chloroplast development remained unknown. Here, we conducted an extensive analysis of the plsp1-null mutant to address the significance of lumenal protein maturation in thylakoid development. Plastids that lack Plsp1 were found to accumulate vesicles of variable sizes in the stroma. Analyses of the mutant plastids revealed that the lack of Plsp1 causes a reduction in accumulation of thylakoid proteins and that Plsp1 is involved in maturation of two additional lumenal proteins, OE23 and plastocyanin. Further immunoblotting and electron microscopy immunolocalization studies showed that OE33 associates with the stromal vesicles of the mutant plastids. Finally, we used a genetic complementation system to demonstrate that accumulation of improperly processed forms of Toc75 in the plastid envelope does not disrupt normal plant development. These results suggest that proper maturation of lumenal proteins may be a key process for correct assembly of thylakoids.Capture of light energy and subsequent electron transport reactions are key steps in photosynthesis. In eukaryotic cells, these reactions occur in thylakoids, the internal membrane systems of chloroplasts. Thylakoid constituents, i.e. membrane lipids, photosynthetic pigments, and various proteins, have been well defined (Schröder and Kieselbach, 2003; Peltier et al., 2004; Benning and Ohta, 2005; DellaPenna and Pogson, 2006; Masuda and Fujita, 2008), and genetic disruptions of their biosynthesis result in a severe reduction of thylakoid development (e.g. Dörmann et al., 1995; Jarvis et al., 1995; Hsieh and Goodman, 2005). Thylakoids depend for their lipid supply on the envelope membranes (Dörmann and Benning, 2002). Early cytological studies suggested that thylakoids derive from the inner envelope membrane by invagination followed by vesicle traffic and/or lateral transfer of membrane constituents (e.g. Mühlethaler and Frey-Wyssling, 1959; von Wettstein, 1959; Hoober et al., 1991). Several putative components for the vesicle trafficking have been identified (Hugueney et al., 1995; Kroll et al., 2001; Wang et al., 2004), although the mode of their functions remains elusive. Overall, molecular details of thylakoid development are not yet fully understood.Proteins found in thylakoid membranes are encoded in either the chloroplast or nuclear genome, whereas lumenal proteins are encoded exclusively in the nucleus (Robinson and Mant, 2005). All of the nuclear-encoded thylakoid proteins identified so far are synthesized on cytosolic ribosomes with N-terminal extensions called transit peptides. These precursor proteins first traverse the double-membrane envelope via the general import machinery called the TOC and TIC complexes (for translocon at the outer- and inner-envelope-membranes of chloroplasts; Schnell et al., 1997), and their transit peptides are removed by a soluble metalloprotease, stromal processing peptidase (Richter et al., 2005). Extensive biochemical and genetic studies have identified four distinct pathways in the stroma to mediate sorting of these nuclear-encoded proteins to thylakoids (Robinson and Mant, 2005; Cline and Dabney-Smith, 2008). The cpSec (for chloroplast Sec) and cpTat (for chloroplast twin-arginine translocation) pathways catalyze protein targeting to the lumen. The cpSRP (for chloroplast signal recognition particle) pathway mediates sorting of the most abundant membrane proteins, LHCPs (for light-harvesting chlorophyll binding proteins), whereas a nonassisted spontaneous pathway targets several other integral membrane proteins in thylakoids. All known nuclear-encoded substrates of cpSec (e.g. OE33 [for oxygen evolving complex subunit 33] and plastocyanin), cpTat (e.g. OE23, OE17, and PsaN), and some spontaneous (e.g. CFoII, PsbW, and PsbX) pathways carry a cleavable thylakoid-targeting signal peptide that follows the stroma-targeting transit peptide in their N termini (Robinson and Mant, 2005). The signal peptides are necessary for the intraorganellar targeting and are removed in the lumen by a type I signal peptidase (SPase I) called thylakoidal processing peptidase (TPP).SPases I are membrane-bound Ser proteases responsible for cleavage of intracellular and/or intraorganellar targeting sequences (signal peptides) from numerous proteins in both prokaryotic and eukaryotic cells (Paetzel et al., 2002). In bacteria, it was shown that signal peptide cleavage is required for some proteins to be released from the plasma membrane during or after their translocation from the cytoplasm (Dalbey and Wickner, 1985), and SPases I are essential for viability (Date, 1983; Cregg et al., 1996; Zhang et al., 1997; Zhbanko et al., 2005). In thylakoids, by contrast, removal of the signal peptide was shown in vivo to be dispensable for the function and assembly of a chloroplast-encoded substrate, cytochrome f, of the green alga Chlamydomonas reinhardtii (Kuras et al., 1995; Baymann et al., 1999). In addition, the precursor form of spinach (Spinacia oleracea) OE33 that contains both the stroma- and thylakoid-targeting sequences was able to reconstitute oxygen evolution activity in vitro (Popelkova et al., 2002). Nonetheless, the importance of signal peptide cleavage for thylakoid biogenesis remains largely unexplored. In 1998, the first plant TPP cDNA was identified from Arabidopsis (Arabidopsis thaliana) seedlings based on similarity of the encoded protein to a cyanobacterial SPase I in its sequence (Chaal et al., 1998). An immunoblot assay showed thylakoid localization of this protein (At2g30440), and an in vitro assay confirmed its processing activity against wheat (Triticum aestivum) OE23 (Chaal et al., 1998). More recent genetic and immunological studies showed that a second TPP isoform in Arabidopsis, named plastidic type I signal peptidase 1 (Plsp1), is involved in processing of an envelope protein, Toc75, and a lumenal protein, OE33 (Inoue et al., 2005; Shipman and Inoue, 2009). The genetic study also showed that Plsp1 is necessary for proper thylakoid development (Inoue et al., 2005), although the mode of its action remains elusive.In this work, we conducted a more extensive characterization of plsp1-1 plastids to demonstrate the significance of protein maturation for thylakoid development. Disruption of the PLSP1 gene led to a reduction in the level of thylakoid proteins and to accumulation of improperly processed forms of multiple lumenal proteins, in addition to those of OE33 and Toc75. Plastids lacking Plsp1 accumulated stroma-localized vesicles of various sizes, at which OE33 was present. Finally, we used a genetic complementation system to show that complete maturation of the envelope protein Toc75 is dispensable for proper plant development. These results suggest that the maturation of lumenal proteins may be necessary for proper development of thylakoids.     

Polygalacturonase causes lygus-like damage on plants: cloning and identification of western tarnished plant bug (Lygus hesperus) polygalacturonases secreted during feeding

Polygalacturonase (PG), an enzyme that degrades pectin within the plant tissue cell wall, has been postulated as the chemical cause of damage to plants by the mirid Lygus hesperus. Micro-injection of two pure recombinant Aspergillus niger PG II protein forms, the wild type enzymically active and the mutant inactive one, into alfalfa (Medicago sativa L.) florets, demonstrates that the enzymatic activity rather than the PG protein structure per se elicits damage symptoms. A PG gene family has been described for the tarnished plant bug, L. lineolaris. Here we report cloning members of the L. hesperus PG gene family, Lhpg2, obtained with L. lineolaris PG-specific primers and a novel Lhpg4, amplified with degenerate primers that were designed based, in part on the N-terminal sequence from an active, partially purified L. hesperus salivary gland PG protein. Proteomic analyses revealed that the salivary gland PGs encoded by Lhpg2 and Lhpg4 are detected in a diet into which L. hesperus has extruded its saliva when feeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: Henryk Czosnek     

Donor variation in the growth properties and osteogenic potential of human marrow stromal cells.

Human marrow stromal cells (MSCs) were isolated from posterior illiac crest marrow aspirates obtained from 17 healthy donors, ages 19-45 years, with no apparent physical disability. First passage hMSCs exhibited growth rates in vitro that varied up to 12-fold between donors. No correlation between growth rate and the age or gender of the donor was evident (P 相似文献   

Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase

Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.     

Additive concentration effects on enantioselective separations in supercritical fluid chromatography

Polar additive concentration effects in supercritical fluid chromatography were studied on chiral stationary phases having either a macrocyclic glycopeptide or a derivatized polysaccharide as the chiral selector. Two basic additives, isopropylamine and triethylamine, were incorporated into the methanol modifier at various concentrations and the effects on retention, selectivity, and resolution were monitored. Many of the analytes failed to elute from the macrocyclic glycopeptide stationary phase in the absence of an additive and the most noticeable effect of increasing additive concentration was a significant decrease in retention. On the derivatized polysaccharide stationary phase the additives had little effect on retention, but they did foster significant improvements in peak shape and resolution.     

Identification of in vivo phosphorylation sites of MLK3 by mass spectrometry and phosphopeptide mapping

MLK3 is a serine/threonine protein kinase that functions as an upstream activator of the JNK pathway. Previous work has suggested that MLK3 is a multiphosphorylated protein. In this study, mass spectrometry coupled with comparative phosphopeptide mapping was used to directly characterize MLK3 in vivo phosphorylation sites. Various types of mass spectrometry were used to analyze MLK3 tryptic peptides separated by C18 reverse-phase HPLC, leading to the identification of Ser(524), Ser(654), Ser(705), Ser(740), Ser(758), Ser(770), Ser(793), and a site found on peptide Ser(11)-Arg(37) within a Gly-rich region as MLK3 phosphorylation sites. Additionally, porous graphitic carbon chromatography successfully retained and resolved phosphopeptides that had eluted along with nonvolatile salts and buffers in the flowthrough fractions from the C18 column. Following resolution by PGC chromatography, MALDI-MS in conjunction with alkaline phosphatase treatment identified Ser(555), Ser(556), Ser(724), and Ser(727) as sites of phosphorylation on MLK3. A proline residue immediately follows 7 of the 11 unambiguously identified phosphorylation sites, suggesting that MLK3 may be a target of proline-directed kinases. Finally, two-dimensional phosphopeptide mapping confirmed that phosphorylation of Ser(555) and Ser(556) of MLK3 is induced by the activated small GTPase Cdc42.     

Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695

We have created early-onset transgenic (Tg) models by exploiting the synergistic effects of familial Alzheimer's disease mutations on amyloid beta-peptide (Abeta) biogenesis. TgCRND8 mice encode a double mutant form of amyloid precursor protein 695 (KM670/671NL+V717F) under the control of the PrP gene promoter. Thioflavine S-positive Abeta amyloid deposits are present at 3 months, with dense-cored plaques and neuritic pathology evident from 5 months of age. TgCRND8 mice exhibit 3,200-4,600 pmol of Abeta42 per g brain at age 6 months, with an excess of Abeta42 over Abeta40. High level production of the pathogenic Abeta42 form of Abeta peptide was associated with an early impairment in TgCRND8 mice in acquisition and learning reversal in the reference memory version of the Morris water maze, present by 3 months of age. Notably, learning impairment in young mice was offset by immunization against Abeta42 (Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T. J., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St. George-Hyslop, P., and Westaway, D. (2000) Nature 408, 979-982). Amyloid deposition in TgCRND8 mice was enhanced by the expression of presenilin 1 transgenes including familial Alzheimer's disease mutations; for mice also expressing a M146L+L286V presenilin 1 transgene, amyloid deposits were apparent by 1 month of age. The Tg mice described here suggest a potential to investigate aspects of Alzheimer's disease pathogenesis, prophylaxis, and therapy within short time frames.