Stage-Specific Changes in Calcium-Regulated Protein Phosphorylation in Developing Tomato Fruits

The role of calcium and calmodulin in the in vitro phosphorylationof soluble and membrane proteins was studied in relation togrowth and development of tomato fruits. Calcium at micromolarconcentrations promoted the phosphorylation of both solubleand membrane proteins. The calmodulin antagonists, chlorpromazineand trifluoperazine, inhibited the phosphorylation of severalproteins. Qualitative changes were observed in the pattern ofprotein phosphorylation at different developmental stages. Therewas a general decrease in protein phosphorylation towards ripening.These results indicated that calcium may be involved in theregulation of phosphorylation of different proteins at differentstages of fruit development. ¹ Scientific Paper No. 7149, College of Agriculture and HomeEconomics, Washington State University, Pullman, Project 0321. ² Supported in part by a grant from the National Science Foundation.DCB-8502215. (Received May 14, 1985; Accepted September 12, 1985)     

Analysis of an osmotically regulated pathogenesis-related osmotin gene promoter

Osmotin is a small (24 kDa), basic, pathogenesis-related protein, that accumulates during adaptation of tobacco (Nicotiana tabacum) cells to osmotic stress. There are more than 10 inducers that activate the osmotin gene in various plant tissues. The osmotin promoter contains several sequences bearing a high degree of similarity to ABRE, as-1 and E-8 cis element sequences. Gel retardation studies indicated the presence of at least two regions in the osmotin promoter that show specific interactions with nuclear factors isolated from cultured cells or leaves. The abundance of these binding factors increased in response to salt, ABA and ethylene. Nuclear factors protected a 35 bp sequence of the promoter from DNase I digestion. Different 5 deletions of the osmotin promoter cloned into a promoter-less GUS-NOS plasmid (pBI 201) were used in transient expression studies with a Biolistic gun. The transient expression studies revealed the presence of three distinct regions in the osmotin promoter. The promoter sequence from –108 to –248 bp is absolutely required for reporter gene activity, followed by a long stretch (up to –1052) of enhancer-like sequence and then a sequence upstream of –1052, which appears to contain negative elements. The responses to ABA, ethylene, salt, desiccation and wounding appear to be associated with the –248 bp sequence of the promoter. This region also contains a putative ABRE (CACTGTG) core element. Activation of the osmotin gene by various inducers is discussed in view of antifungal activity of the osmotin protein.     

The effect of iron on the primary root elongation of Arabidopsis during phosphate deficiency

Root architecture differences have been linked to the survival of plants on phosphate (P)-deficient soils, as well as to the improved yields of P-efficient crop cultivars. To understand how these differences arise, we have studied the root architectures of P-deficient Arabidopsis (Arabidopsis thaliana Columbia-0) plants. A striking aspect of the root architecture of these plants is that their primary root elongation is inhibited when grown on P-deficient medium. Here, we present evidence suggesting that this inhibition is a result of iron (Fe) toxicity. When the Fe concentration in P-deficient medium is reduced, we observe elongation of the primary root without an increase in P availability or a corresponding change in the expression of P deficiency-regulated genes. Recovery of the primary root elongation is associated with larger plant weights, improved ability to take up P from the medium, and increased tissue P content. This suggests that manipulating Fe availability to a plant could be a valuable strategy for improving a plant's ability to tolerate P deficiency.     

Identification and properties of anti-chaperone-like peptides derived from oxidized bovine lens betaL-crystallins

Thermal aggregation of betaL-crystallin was higher in the presence of peptide fragments generated from oxidized and trypsin-digested betaL-crystallin compared with thermal aggregation of the control proteins without oxidized betaL-crystallin fragments. Increased aggregation of betaL-crystallin was also observed despite the presence of alpha-crystallin (which has anti-aggregating properties) in the system. Self-aggregation of the oxidized betaL-crystallin fragments per se was not observed under the experimental conditions. Reverse-phase HPLC analysis of the precipitate obtained after heating a mixture of betaL-crystallin and oxidized betaL-crystallin fragments revealed that more than one peptide co-precipitates with betaL-crystallin. Electrospray mass spectrometry analysis of the peptides revealed that the molecular weight(s) of the peptides ranged from 1400-1800. Tandem mass spectrometry and a data base search revealed that two of the peptides originated from betaA4-crystallin (LTIFEQENFLGR, residues 121-132) and betaB3-crystallin (AINGTWVGYEFPGYR, residues 153-167) respectively. Oxidized synthetic peptides representing the same sequence were also found to enhance the aggregation of betaL-crystallin in a manner similar to oxidized lens betaL-crystallin peptides. These data suggest that the polypeptides generated after oxidation and proteolysis of betaL-crystallins interact with denaturing proteins and facilitate their aggregation and light scattering, thus behaving like anti-chaperones.     

Two novel hexadepsipeptides with several modified amino acid residues isolated from the fungus Isaria

Two new cyclohexadepsipeptides have been isolated from the fungus Isaria. Fungal growth in solid media yielded hyphal strands from which peptide fractions were readily isolable by organic-solvent extraction. Two novel cyclodepsipeptides, isaridin A and isaridin B, have been isolated by reverse-phase HPLC, and characterized by ESI-MS and 1H-NMR. Single crystals of both peptides have been obtained, and their 3D structures were elucidated by X-ray diffraction. The isaridins contain several unusual amino acid residues. The sequences are cyclo(beta-Gly-HyLeu-Pro-Phe-NMeVal-NMePhe) and cyclo(beta-Gly-HyLeu-beta-MePro-Phe-NMeVal-NMePhe), where NMeVal is N-methylvaline, NMePhe N-methylphenylalanine, and HyLeu hydroxyleucine (= 2-hydroxy-4-methylpentanoic acid). The two peptides differ from one another at residue 3, isaridin A having an (S)-proline at this position, while beta-methyl-(S)-proline (= (2S,3S)-2,3,4,5-tetrahydro-3-methyl-1H-pyrrole-2-carboxylic acid) is found in isaridin B. The solid-state conformations of both cyclic depsipeptides are characterized by the presence of two cis peptide bonds at HyLeu(2)-Pro(3)/HyLeu(2)-beta-MePro(3) and NMeVal(5)-NMePhe(6), respectively. In isaridin A, a strong intramolecular H-bond is observed between Phe(4)CO...HNbeta-Gly(1), and a similar, but weaker, interaction is observed between beta-Gly(1)CO...HNPhe(4). In contrast, in isaridin B, only a single intramolecular H-bond is observed between beta-Gly(1)CO...HNPhe(4).     

Clostridium thermocellum Xyn10B carbohydrate-binding module 22-2: the role of conserved amino acids in ligand binding

The majority of plant cell wall hydrolases are modular enzymes which, in addition to a catalytic module, possess one or more carbohydrate-binding modules (CBMs). These carbohydrate-active enzymes and their constituent modules have been classified into a number of families based upon amino acid sequence similarity. The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that belong to family 22 (CBM22). The crystal structure of the C-terminal CBM22 (CBM22-2) was determined in a previous study [Charnock, S. J., et al. (2000) Biochemistry 39, 5013--5021] and revealed a surface cleft which presents several conserved residues that are implicated in ligand binding. These amino acids have been substituted and the structure and biochemical properties of the mutants analyzed. The data show that R25A, W53A, Y103A, Y136A, and E138A exhibit greatly reduced affinity for xylotetraose relative to that of the wild-type protein. Conversely, mutations Y103F and Y136F have little effect on ligand binding. Using thermodynamic, X-ray, and NMR measurements on the mutants, we show that the cleft of CBM22-2 does indeed form the ligand-binding site. Trp 53 and Tyr 103 most likely participate in hydrophobic stacking interactions with the ligand, while Glu 138 makes one or more important hydrogen bonds with the tetrasaccharide. Although Arg 25 and Tyr 136 are likely to form hydrogen bonds with the ligand, they are also shown to play a critical role in maintaining the structural integrity of the binding cleft.