Structural basis for thermostability of endo-1,5-alpha-L-arabinanase from Bacillus thermodenitrificans TS-3

The crystal structure of a thermostable endo-1,5-alpha-L-arabinanase, ABN-TS, from Bacillus thermodenitrificans TS-3 was determined at 1.9 A to an R-factor of 18.3% and an R-free-factor of 22.5%. The enzyme molecule has a five-bladed beta-propeller fold. The substrate-binding cleft formed across one face of the propeller is open on both sides to allow random binding of several sugar units in the polymeric substrate arabinan. The beta-propeller fold is stabilized through a ring closure. ABN-TS exhibits a new closure-mode involving residues in the N-terminal region: Phe7 to Gly21 exhibit hydrogen bonds and hydrophobic interactions with the first and last blades, and Phe4 links the second and third blades through a hydrogen bond and an aromatic stacking interaction, respectively. The role of the N-terminal region in the thermostability was confirmed with a mutant lacking 16 amino acid residues from the N-terminus of ABN-TS.     

Interaction of plastocyanin with oligopeptides: effect of lysine distribution within the peptide

We synthesized and purified four oligopeptides containing four lysines (KKKK, GKKGGKK, KKGGGKK, and KGKGKGK) as models for the plastocyanin (PC) interacting site of cytochrome f. These peptides competitively inhibited electron transfer between cytochrome c and PC. The inhibitory effect increased as the peptide concentrations were increased. The association constants between PC and the peptides did not differ significantly (3500-5100 M(-1)), although the association constant of PC-KGKGKGK was a little larger than the constants between PC and other peptides. Changes in the absorption spectrum of PC were observed when the peptides were added to the PC solution: peaks and troughs were detected at about 460 and 630 nm and at about 560 and 700 nm, respectively, in the difference absorption spectra between the spectra with and without peptides. These changes were attributed to the structural change at the copper site of PC by interaction with the peptides. The structural change was most significant when tetralysine was used. These results show that binding of the oligopeptide to PC is slightly more efficient when lysines are distributed uniformly within the peptide, whereas the structural change of PC becomes larger when the lysines are close to each other within the peptide.     

Elucidation of salt-tolerance metabolic pathways in contrasting rice genotypes and their segregating progenies

Key message

Differentially expressed antioxidant enzymes, amino acids and proteins in contrasting rice genotypes, and co-location of their genes in the QTLs mapped using bi-parental population, indicated their role in salt tolerance.

Abstract

Soil salinity is a major environmental constraint limiting rice productivity. Salt-tolerant ‘CSR27’, salt-sensitive ‘MI48’and their extreme tolerant and sensitive recombinant inbred line (RIL) progenies were used for the elucidation of salt stress tolerance metabolic pathways. Salt stress-mediated biochemical and molecular changes were analyzed in the two parents along with bulked-tolerant (BT) and bulked-sensitive (BS) extreme RILs. The tolerant parent and BT RILs suffered much lower reduction in the chlorophyll as compared to their sensitive counterparts. Activities of antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD) and non-enzymatic antioxidant ascorbic acid were much higher in salt-stressed CSR27 and BT RILs than MI48 and BS RILs. Further, the tolerant lines showed significant enhancement in the levels of amino acids methionine and proline in response to salt stress in comparison to the sensitive lines. Similarly, the tolerant genotypes showed minimal reduction in cysteine content whereas sensitive genotypes showed a sharp reduction. Real time PCR analysis confirmed the induction of methionine biosynthetic pathway (MBP) enzymes cystathionine-β synthase (CbS), S-adenosyl methionine synthase (SAMS), S-adenosyl methionine decarboxylase (SAMDC) and serine hydroxymethyl transferase (SHMT) genes in tolerant lines, suggesting potential role of the MBP in conferring salt tolerance in rice variety CSR27. Proteome profiling also confirmed higher expression of SOD, POD and plastidic CbS and other proteins in the tolerant lines, whose genes were co-located in the QTL intervals for salt tolerance mapped in the RIL population. The study signifies integrated biochemical-molecular approach for identifying salt tolerance genes for genetic improvement for stress tolerant rice varieties.

     

Formation of the nuclear envelope during mitotic anaphase inSpirogyra

The formation of the nuclear envelope in the mitosis ofSpirogyra was studied with an electron microscope. The nuclear envelope was disrupted around the spindle equator in the metaphase. Many small vesicles were observed in the metaphase spindle. These vesicles surrounded the masses of chromosomes and nucleolar substance in the early anaphase, and they fused with each other to form daughter nuclear envelopes during the early anaphase. The formation of new envelopes from small vesicles at such an early mitotic anaphase is reported here for the first time. The possible origin of these vesicles is also discussed.     

Sequencing and Expression of the Gene that Encodes a 20-kDa Polypeptide of the PSI Complex from Cucumber Cotyledon

A cDNA clone encoding the polypeptide from cucumber PS I thatmigrates with an apparent molecular weight of 20 kDa on SDS-polyacrylamidegels has been isolated. The 907-bp sequence of this clone hasbeen determined and contains one large open reading frame thatencodes a 22,720-Da precursor polypeptide (207 amino acid residues).The molecular weight of the mature polypeptide was predictedto be 17,037-Da (153 amino acid residues). The deduced aminoacid sequence of this protein indicates that it is routed towardsthe stromal side of the thylakoid membrane and has no membrane-spanningregions. The sequence also confirmed the identity of the proteinas the product of the psa D gene. Chemical cross-linking offerredoxin to the PS I complex identified the 20-kDa subunitas the ferredoxin-binding protein. Northern hybridization experimentsrevealed that the mRNA of approximately 1,100 nucleotides forthe 20-kDa polypeptide was present in etiolated cucumber cotyledons,and its level increased about 5-fold during greening. The 20-kDapolypeptide was not detected by immunoblotting in etiolatedcotyledons, and it accumulated only after illumination. Labelingexperiments in vivo showed the absence of incorporation of [³⁵S]Metinto the polypeptide in etiolated cotyledons. These resultssuggest that the expression of the psa D gene is controlledat the translational level. (Received April 5, 1990; Accepted June 28, 1990)     

Electron--nuclear-double-resonance studies of spinach plastocyanin.

Electron--nuclear-double-resonance (ENDOR) studies of spinach plastocyanin revealed that 14N-hyperfine couplings from its two co-ordinated imidazole groups are characteristically small in anisotropy and relatively high in ENDOR frequencies and that the two imidazole groups are magnetically equivalent. The relatively strong copper-imidazole co-ordinate bonds of plastocyanin are interpreted to be a result of the coexistence of a strong copper--thiol co-ordinate bond.     

Correlation between number of placental detachments and amount of postpartum uterine involution in mice

Both the uterine weight and total hydroxyproline content augumented in parallel with the number of placental scars on the day of parturition. In the uterus having any number of placental scars, the time of half loss of the organ weight and the total hydroxyproline content, respectively, was the same. These results suggested the amount of postpartum involution increases as the number of placental detachment becomes high.     

Suppressed expression of the apoplastic ascorbate oxidase gene increases salt tolerance in tobacco and Arabidopsis plants

Transgenic tobacco plants expressing the ascorbate oxidase (AAO) gene in sense and antisense orientations, and an Arabidopsis mutant in which the T-DNA was inserted into a putative AAO gene, were used to examine the potential roles of AAO for salt-stress tolerance in plants. AAO activities in the transgenic tobacco plants expressing the gene in sense and antisense orientations were, respectively, about 16-fold and 0.2-fold of those in the wild type. Under normal growth conditions, no significant differences in phenotypes were observed, except for a delay in flowering time in the antisense plants. However, at high salinity, the percentage germination, photosynthetic activity, and seed yields were higher in antisense plants, with progressively lower levels in the wild type and the sense plants. The redox state of apoplastic ascorbate in sense plants was very low even under normal growth conditions. Upon salt stress, the redox state of symplastic and apoplastic ascorbate decreased among the three types of plants, but was lowest in the sense plants. The hydrogen peroxide contents in the symplastic and apoplastic spaces were higher in sense plants, progressively lower in the wild type, followed by the antisense plants. The Arabidopsis T-DNA inserted mutant exhibited very low ascorbate oxidase activity, and its phenotype was similar to that of antisense tobacco plants. These results suggest that the suppressed expression of apoplastic AAO under salt-stress conditions leads to a relatively low level of hydrogen peroxide accumulation and a high redox state of symplastic and apoplastic ascorbate which, in turn, permits a higher seed yield.     

Metabolic engineering for betaine accumulation in microbes and plants

Plants accumulate a variety of osmoprotectants that improve their ability to combat abiotic stresses. Among them, betaine appears to play an important role in conferring resistance to stresses. Betaine is synthesized via either choline oxidation or glycine methylation. An increased betaine level in transgenic plants is one of the potential strategies to generate stress-tolerant crop plants. Here, we showed that an exogenous supply of serine or glycine to a halotolerant cyanobacterium Aphanothece halophytica, which synthesizes betaine from glycine by a three-step methylation, elevated intracellular accumulation of betaine under salt stress. The gene encoding 3-phosphoglycerate dehydrogenase (PGDH), which catalyzes the first step of the phosphorylated pathway of serine biosynthesis, was isolated from A. halophytica. Expression of the Aphanothece PGDH gene in Escherichia coli caused an increase in levels of betaine as well as glycine and serine. Expression of the Aphanothece PGDH gene in Arabidopsis plants, in which the betaine synthetic pathway was introduced via glycine methylation, further increased betaine levels and improved the stress tolerance. These results demonstrate that PGDH enhances the levels of betaine by providing the precursor serine for both choline oxidation and glycine methylation pathways.