BLUE AND WHITE LIGHT EFFECTS ON CHLOROPLAST DEVELOPMENT IN A SOYBEAN MUTANT

Phenotypic difference for chloroplast development between the normal green (CL1) and the Cy₉y₉ soybean mutant was observed when the plants were grown under 18W m^?2 white or blue light. Under these conditions the mutant soybean accumulated less Chi b, neoxanthin, carotene and less total pigment than the CL1 genotype. Chloroplasts of the Cy₉y₉ line were deficient in the LHP complex relative to that of chloroplasts from the normal soybean. Specific differences were noted between chloroplasts from plants grown under blue and white light. Accumulations of a 34 kD (PSII) and a 16–17 kD (PSI) membrane polypeptide were decreased by blue light in both soybean genotypes. Blue light induced a greater accumulation of a 32 kD (PSII) polypeptide than white light. Blue light reduced granal thylakoid stacking and increased the proportion of stroma thylakoids compared to those that developed under white light. PSI electron transport activity was stimulated by the blue light treatment more than that of PSII.     

Crystal Structures of Bacterial (6‐4) Photolyase Mutants with Impaired DNA Repair Activity

PhrB from Agrobacterium fabrum is the first prokaryotic photolyase which repairs (6‐4) UV DNA photoproducts. The protein harbors three cofactors: the enzymatically active FAD chromophore, a second chromophore, 6,7‐dimethyl‐8‐ribityllumazine (DMRL) and a cubane‐type Fe‐S cluster. Tyr424 of PhrB is part of the DNA‐binding site and could provide an electron link to the Fe‐S cluster. The PhrB_Y424F mutant showed reduced binding of lesion DNA and loss of DNA repair. The mutant PhrB_I51W is characterized by the loss of the DMRL chromophore, reduced photoreduction and reduced DNA repair capacity. We have determined the crystal structures of both mutants and found that both mutations only affect local protein environments, whereas the overall fold remained unchanged. The crystal structure of PhrB_Y424F revealed a water network extending to His366, which are part of the lesion‐binding site. The crystal structure of PhrB_I51W shows how the bulky Trp leads to structural rearrangements in the DMRL chromophore pocket. Spectral characterizations of PhrB_I51W suggest that DMRL serves as an antenna chromophore for photoreduction and DNA repair in the wild type. The energy transfer from DMRL to FAD could represent a phylogenetically ancient process.     

Structure‐Guided Minimalist Redesign of the L‐Fuculose‐1‐Phosphate Aldolase Active Site: Expedient Synthesis of Novel Polyhydroxylated Pyrrolizidines and their Inhibitory Properties Against Glycosidases and Intestinal Disaccharidases

A minimalist active site redesign of the L ‐fuculose‐1‐phosphate aldolase from E. coli FucA was envisaged, to extend its tolerance towards bulky and conformationally restricted N‐Cbz‐amino aldehyde acceptor substrates (Cbz=benzyloxycarbonyl). Various mutants at the active site of the FucA wild type were obtained and screened with seven sterically demanding N‐Cbz‐amino aldehydes including N‐Cbz‐prolinal derivatives. FucA F131A showed an aldol activity of 62 μmol h^?1 mg^?1 with (R)‐N‐Cbz‐prolinal, whereas no detectable activity was observed with the FucA wild type. For the other substrates, the F131A mutant gave aldol activities from 4 to about 25 times higher than those observed with the FucA wild type. With regard to the stereochemistry of the reactions, the (R)‐amino aldehydes gave exclusively the anti configured aldol adducts whereas their S counterparts gave variable ratios of anti/syn diastereoisomers. Interestingly, the F131A mutant was highly stereoselective both with (R)‐ and with (S)‐N‐Cbz‐prolinal, exclusively producing the anti and syn aldol adducts, respectively. Molecular models suggest that this improved activity towards bulky and more rigid substrates, such as N‐Cbz‐prolinal, could arise from a better fit of the substrate into the hydrophobic pocket created by the F131A mutation, due to an additional π–cation interaction with the residue K205′ and to efficient contact between the substrate and the mechanistically important Y113′ and Y209′ residues. An expedient synthesis of novel polyhydroxylated pyrrolizidines related to the hyacinthacine and alexine types was accomplished through aldol additions of dihydroxyacetone phosphate (DHAP) to hydroxyprolinal derivatives with the hyperactive FucA F131A as catalyst. The iminocyclitols obtained were fully characterised and found to be moderate to weak inhibitors (relative to 1,4‐dideoxy‐1,4‐imino‐L ‐arabinitol (LAB) and 1,4‐dideoxy‐1,4‐imino‐D ‐arabinitol (DAB)) against glycosidases and rat intestinal saccharidases.     

Engineered C–N Lyase: Enantioselective Synthesis of Chiral Synthons for Artificial Dipeptide Sweeteners

Aspartic acid derivatives with branched N‐alkyl or N‐arylalkyl substituents are valuable precursors to artificial dipeptide sweeteners such as neotame and advantame. The development of a biocatalyst to synthesize these compounds in a single asymmetric step is an as yet unmet challenge. Reported here is an enantioselective biocatalytic synthesis of various difficult N‐substituted aspartic acids, including N‐(3,3‐dimethylbutyl)‐l ‐aspartic acid and N‐[3‐(3‐hydroxy‐4‐methoxyphenyl)propyl]‐l ‐aspartic acid, precursors to neotame and advantame, respectively, using an engineered variant of ethylenediamine‐N,N′‐disuccinic acid (EDDS) lyase from Chelativorans sp. BNC1. This engineered C–N lyase (mutant D290M/Y320M) displayed a remarkable 1140‐fold increase in activity for the selective hydroamination of fumarate compared to that of the wild‐type enzyme. These results present new opportunities to develop practical multienzymatic processes for the more sustainable and step‐economic synthesis of an important class of food additives.     

Targeted Inactivation of DNA Photolyase Genes in Medaka Fish (Oryzias latipes)

Proteins of the cryptochrome/photolyase family (CPF) exhibit sequence and structural conservation, but their functions are divergent. Photolyase is a DNA repair enzyme that catalyzes the light‐dependent repair of ultraviolet (UV)‐induced photoproducts, whereas cryptochrome acts as a photoreceptor or circadian clock protein. Two types of DNA photolyase exist: CPD photolyase, which repairs cyclobutane pyrimidine dimers (CPDs), and 6‐4 photolyase, which repairs 6‐4 pyrimidine–pyrimidone photoproducts (6‐4PPs). Although the Cry‐DASH protein is classified as a cryptochrome, it also has light‐dependent DNA repair activity. To determine the significance of the three light‐dependent repair enzymes in recovering from solar UV‐induced DNA damage at the organismal level, we generated mutants in each gene in medaka using the CRISPR genome editing technique. The light‐dependent repair activity of the mutants was examined in vitro in cultured cells and in vivo in skin tissue. Light‐dependent repair of CPD was lost in the CPD photolyase‐deficient mutant, whereas weak repair activity against 6‐4PPs persisted in the 6‐4 photolyase‐deficient mutant. These results suggest the existence of a heretofore unknown 6‐4PP repair pathway and thus improve our understanding of the mechanisms of defense against solar UV in vertebrates.     

Photoinhibition in vivo and in vitro involves weakly coupled chlorophyll-protein complexes

In the present study the analysis of the relation between the excited state population in the photosystem II (PSII) antenna and photoinactivation has been extended from an in vitro system, isolated thylakoids, to an in vivo system, Chlamydomonas reinhardtii cells. The results indicate that the excited state quenching by an added singlet quencher induces maximal protection against photoinhibition of about 30% of that expected on the basis of the observed light intensity-treatment time reciprocity rule. Similar results, obtained previously with thylakoids, have been interpreted in terms of damaged or incorrectly assembled complexes that play an important role in photoinhibition in the thylakoid membranes (Santabarbara, S., K. Neverov, F. M. Garlaschi, G. Zucchelli and R. C. Jennings [2001] Involvement of uncoupled antenna chlorophylls in photoinhibition in thylakoids. FEBS Lett. 491, 109-113.). In an attempt to better define this aspect, the photoinhibition action spectra were determined for mutant barley thylakoids, lacking the chlorophyll (Chl) a-b complexes of the outer antenna, and for its wild type. The results indicate that in both systems the action spectra are significantly blueshifted (2-4 nm) and are broader than the PSII absorption in the membranes. These data are interpreted in terms of a heterogeneous population of outer and inner antenna pigment-protein complexes that contain significant levels of uncoupled Chl.     

Comparative Time-Resolved Photosystem II Chlorophyll a Fluorescence Analyses Reveal Distinctive Differences between Photoinhibitory Reaction Center Damage and Xanthophyll Cycle-Dependent Energy Dissipation*

Abstract— The photosystem II (PSII) reaction center in higher plants is susceptible to photoinhibitory molecular damage of its component pigments and proteins upon prolonged exposure to excess light in air. Higher plants have a limited capacity to avoid such damage through dissipation, as heat, of excess absorbed light energy in the PSII light-harvesting antenna. The most important pho-toprotective heat dissipation mechanism, induced under excess light conditions, includes a concerted effect of the trans-thylakoid pH gradient (ΔpH) and the carotenoid pigment interconversions of the xanthophyll cycle. Co-incidentally, both the photoprotective mechanism and photoinhibitory PSII damage decrease the PSII chlorophyll a (Chi a) fluorescence yield. In this paper we present a comparative fluorescence lifetime analysis of the xanthophyll cycle- and photoinhibition-dependent changes in PSII Chi a fluorescence. We analyze multifrequency phase and modulation data using both multicomponent exponential and bimodal Lorentzian fluorescence lifetime distribution models; further, the lifetime data were obtained in parallel with the steady-state fluorescence intensity. The photoinhi-bition was characterized by a progressive decrease in the center of the main fluorescence lifetime distribution from ～2 ns to ～0.5 ns after 90 min of high light exposure. The damaging effects were consistent with an increased nonra-diative decay path for the charge-separated state of the PSII reaction center. In contrast, the ΔpH and xanthophyll cycle had concerted minor and major effects, respectively, on the PSII fluorescence lifetimes and intensity (Gilmore et ah, 1996, Photosynth. Res., in press). The minor change decreased both the width and lifetime center of the longest lifetime distribution; we suggest that this change is associated with the ΔpH-induced activation step, needed for binding of the deepoxidized xanthophyll cycle pigments. The major change increased the fractional intensity of a short lifetime distribution at the expense of a longer lifetime distribution; we suggest that this change is related to the concentration-dependent binding of the deepoxidized xanthophylls in the PSII inner antenna. Further, both the photoinhibition and xanthophyll cycle mechanisms had different effects on the relationship between the fluorescence lifetimes and intensity. The observed differences between the xanthophyll cycle and photoinhibition mechanisms confirm and extend our current basic model of PSII exciton dynamics, structure and function.     

Effectively controlled microfluidic trap for spatiotemporal analysis of the electrotaxis of Caenorhabditis elegans

C. elegans is a popular model organism with a well‐developed neural network. Approximately 60% of the genes in C. elegans have genomic counterparts in humans, including those involved in building neural circuits. Therefore, we can extend the study of human neural network mechanisms to C. elegans which is easy to genetically manipulate. C. elegans shows behavioural responses to various external physical and chemical stimuli. Electrotaxis is one of its distinct behavioural responses, which is defined as movement towards the cathode in an electric field. In this study, we developed an effective microfluidic trap system for analysing electrotaxis in C. elegans. In addition, two mutant strains (unc‐54(s74) and unc‐6(e78)) from wild‐type (N2) worms were screened using the system. Wild‐type (N2) worms and the two mutant strains clearly showed different behavioural responses to the applied electric field, thus enabling the effective screening of the mutant worms from the wild type (N2). This microfluidic system can be utilized as a platform for the study of behavioural responses, and for the sorting and mutant screening of C. elegans.     

The CO Photodissociation and Recombination Dynamics of the W172Y/F282T Ligand Channel Mutant of Rhodobacter sphaeroides aa3 Cytochrome c Oxidase

In the ligand channel of the cytochrome c oxidase from Rhodobacter sphaeroides (Rs aa₃) W172 and F282 have been proposed to generate a constriction that may slow ligand access to and from the active site. To explore this issue, the tryptophan and phenylalanine residues in Rs aa₃ were mutated to the less bulky tyrosine and threonine residues, respectively, which occupy these sites in Thermus thermophilus (Tt) ba₃ cytochrome oxidase. The CO photolysis and recombination dynamics of the reduced wild‐type Rs aa₃ and the W172Y/F282T mutant were investigated using time‐resolved optical absorption spectroscopy. The spectral changes associated with the multiple processes are attributed to different conformers. The major CO recombination process (44 μs) in the W172Y/F282T mutant is ~500 times faster than the predominant CO recombination process in the wild‐type enzyme (~23 ms). Classical dynamic simulations of the wild‐type enzyme and double mutant showed significant structural changes at the active site in the mutant, including movement of the heme a₃ ring‐D propionate toward Cu_B and reduced binuclear center cavity volume. These structural changes effectively close the ligand exit pathway from the binuclear center, providing a basis for the faster CO recombination in the double mutant.     

Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base‐Modified Nucleosides

A wide range of natural purine analogues was used as probe to assess the mechanism of recognition by the wild‐type (WT) E. coli purine nucleoside phosphorylase (PNP) versus its Ser90Ala mutant. The results were analyzed from viewpoint of the role of the Ser90 residue and the structural features of the bases. It was found that the Ser90 residue of the PNP 1) plays an important role in the binding and activation of 8‐aza‐7‐deazapurines in the synthesis of their nucleosides, 2) participates in the binding of α‐D ‐pentofuranose‐1‐phosphates at the catalytic site of the PNP, and 3) catalyzes the dephosphorylation of intermediary formed 2‐deoxy‐α‐D ‐ribofuranose‐1‐phosphate in the trans‐2‐deoxyribosylation reaction. 5‐Aza‐7‐deazaguanine manifested excellent substrate activity for both enzymes, 8‐amino‐7‐thiaguanine and 2‐aminobenzothiazole showed no substrate activity for both enzymes. On the contrary, the 2‐amino derivatives of benzimidazole and benzoxazole are substrates and are converted into the N1‐ and unusual N2‐glycosides, respectively. 9‐Deaza‐5‐iodoxanthine showed moderate inhibitory activity of the WT E. coli PNP, whereas 9‐deazaxanthine and its 2′‐deoxyriboside are weak inhibitors.     

The Growth‐promoting Mechanism of Unusual Spectroscopic Form of LH2 (LH4) from Rhodopseudomonas palustris CGA009 in Low Light

The experimental evidence for the growth‐promoting mechanism and the efficiency of energy transfer (EET) of LH4 under low light are still not available. To elucidate the light adaption mechanism of LH4, we deleted the genes pucBAd involved in the synthesis of the α/β polypeptides of LH4 in Rhodopseudomonas palustris CGA009. Compared to wild strain, the growth rate of pucBAd mutant significantly decreased under low light, while there were no significant changes in the growth rate, the contents and compositions of photopigments, absorption spectra of cell lysates under high light. Moreover, the fluorescence quantum efficiency (FQE) was used to further compare the EET between LH2 and LH4. The FQE in LH4 increased up to 1.5‐fold than did in LH2. Collectively, this study showed that LH4 could provide more and high energetic state photons for promoting bacterial phototrophic growth in response to low‐light environment.     

Dual selective nitration in Arabidopsis: Almost exclusive nitration of PsbO and PsbP,and highly susceptible nitration of four non‐PSII proteins,including peroxiredoxin II E

Protein tyrosine nitration is a selective process, as revealed in studies of animals. However, evidence for selective protein nitration in plants is scarce. In this study, Arabidopsis plants were exposed to air with or without nitrogen dioxide at 40 ppm for 8 h in light. Proteins extracted from whole leaves or isolated chloroplasts were subjected to 2D PAGE followed by SYPRO Ruby staining and immunoblotting using an anti‐3‐nitrotyrosine antibody. We determined the relative intensity of a spot on an immunoblot (designated RISI), and relative intensity of the corresponding spot on SYPRO Ruby gel (designated RISS). Proteins that exhibited a high RISI value and/or a high RISI/RISS ratio were considered selectively nitrated. In whole leaf proteins from exposed plants, all immunopositive spots were identified as PsbO1, PsbO2 or PsbP1 by PMF. Thus, nitration was exclusive to PsbO and PsbP, extrinsic proteins of photosystem II (PSII). Their RISI/RISS ratio was ≤1.5. Non‐exposed plants showed very faint nitration. In purified chloroplast proteins, PsbO and PsbP accounted for >80% of the total RISI values, while four non‐PSII proteins, including peroxiredoxin II E, exhibited high RISI/RISS ratios (2.5～6.6). Tyr⁹ of PsbO1 was identified as a nitration site. Thus, nitration is selective for two PSII and four non‐PSII proteins in Arabidopsis.     

DNA Photodamage, Repair, Gene Induction and Genotoxicity Following Exposures to 254 nm UV and 8-Methoxypsoralen Plus UVA in a Eukaryotic Cell System

The induction and repair of different types of photodamage and photogenotoxicity in eukaryotic cells have been the subject of many studies. Little is known about possible links between these phenomena and the induction of DNA damage-inducible genes. We explored this relationship using the yeast Saccharomyces cerevisiae, a pertinent eukaryotic model. Previous results showed that the photogenotoxic potential of 8-methoxypsoralen (8-MOP) plus UVA is higher than that of UV (254 nm). Moreover, the induction of the ribonucleotide reductase gene RNR2 by UV and 8-MOP plus UVA in an RNR2-LACZ fusion strain and the formation of DNA double-strand breaks (dsb) as repair intermediates after such treatments suggest that the latter process could involve a signal for gene induction. To further substantiate this, we measured the induction of the DNA repair gene RAD51 in RAD51-LACZ fusion strains using the dsb repair and recombination deficient mutant rad52 and the corresponding wild type, and we determined the formation of dsb by pulsed-field gel electrophoresis. After treatments, the resealing of dsb formed as repair intermediates was impaired in the rad52 mutant. At equal doses, i.e. the same number of lesions, the induction of the RAD51 gene by UV or 8-MOP plus UVA was significantly reduced in the rad52 mutant as compared with the wild type. The same was true when equitoxic doses were used. Thus, the RAD52 repair pathway appears to play an important role not only in dsb repair but also in gene induction. Furthermore, the signaling pathways initiated by DNA damage and its processing are somewhat linked to the photogenotoxic response.     

Period-four Modulation of Photosystem II Primary Quinone Acceptor (QA) Reduction/Oxidation Kinetics in Thylakoid Membranes

Photosystem II (PSII), a multiprotein complex mainly coded by the chloroplast genome in higher plants and algae, contains the oxygen-evolving complex with four manganese atoms responsible for the oxidation of water. After each absorption of a light quantum by pigment molecules in the light harvesting complexes of PSII, the Mn cluster advances in its oxidation states denoted from S₀ to S₄. The S₄ state decays to S₀ in the dark with the concurrent release of molecular oxygen. Therefore, the oxygen production in PSII exposed to successive single turnover excitations follows a period-four oscillation pattern. The intensity of chlorophyll a fluorescence of PSII is also known to be influenced by the oxidation state of the Mn cluster. In the present work, fluorescence induction kinetics was measured in isolated thylakoids with various initial S-state populations settled by preflashes. The shape of the fluorescence induction traces was strongly affected by preflashes. O-J and J-I phases of the induction followed a period-four oscillation pattern. The results indicate that these changes reflect the influence of the oxidation rate of the Mn cluster on the reduction/oxidation kinetics of the primary quinone acceptor (Q_A) of PSII.     

Phosphotransacetylase as a key factor in biological production of polyhydroxybutyrate

Phosphotransa cetylase (Pta) catalyzes the reversible conversion of, acetyl-coenzyme A (CoA) to acetyl phosphate. Polyhydroxybutyrate (PHB) synthase and accumulation were compared between a Pta-deficient mutant and the wild-type Escherichia coli, which were transformed with pAE100, coding for 3-ketothiolase, NADPH-dependenta cetoacetyl-CoA reductase, and PHB synthase from Ralstonia eutropha. During the growth period, PHB synthase activity in the Pta-deficient mutant was lower than that in the wild type. PHB accumulation in the Pta-deficient mutant, however, was higher than that in wild-type cells grown in Luria-Bertani (LB) medium containing 1% glucose (high C:N ratio). The Pta-deficient mutant showed PHB accumulation even in LB medium (low C:N ratio), whereas wild-type cells showed no PHB accumulation. These data suggest the activation of PHB synthase by acetyl phosphate that is synthesized by Pta. A decrease in Pta activity probably causes some increase in acetyl-CoA as substrate for the PHB synthesis pathway, resulting in increased PHB accumulation.     

Design and Synthesis of Aminoglycoside Antibiotics to Selectively Target 16S Ribosomal RNA Position 1408

The glucopyranosyl moiety (ring I) of paromomycin was modified in a search for novel aminoglycoside antibiotics. The key intermediates were the 4′,6′‐O‐benzylidenated N‐Boc derivative 3 and the azido analogue 18 . The bromobenzoates 4 and 19 were prepared by treating the benzylidene acetals 3 and 18 , respectively, with N‐bromosuccinimide (NBS), and the diol 8 was obtained by hydrogenolysis of 3. The C(6′)‐deoxy derivative 5 was obtained from 4 by treatment with Bu₃SnH. Selective fluorodehydroxylation of 8 gave the fluoro derivative 9. The pseudotrisaccharide 13 was obtained by reductive fragmentaion of the iodo compound 12 obtained from the bromobenzoate 4 . The 3′,6′‐anhydro derivative 20 was obtained upon deacetylation of 19. Standard deprotection gave the C(6′)‐deoxy compound 7 , the fluoro compound 11 , the pseudotrisaccharide 15 , and the 3′,6′‐anhydro‐paromomycin 22 . As compared to paromomycin, the C(6′)‐deoxy and fluorodeoxy derivatives 7 and 11 showed a lower activity against both wild type 1408A and 1408G mutant ribosomes. A lower activity was also found for the 3′,6′‐anhydro derivative 22 and for the pseudotrisaccharide 15 .     

Psb30 is a photosystem II reaction center subunit and is required for optimal growth in high light in Chlamydomonas reinhardtii

The psb30 (ycf12) gene is conserved in a wide variety of oxygenic-photosynthetic organisms except angiosperms and some marine cyanobacteria. Psb30 protein is found in cyanobacterial photosystem II (PSII) core complexes and is dispensable for PSII structure and function. The most recent three-dimensional structure of cyanobacterial PSII core complex has revealed that Psb30 is located in proximity of PsbJ, PsbK, and PsbZ. However Psb30 has not yet been detected in PSII complexes from eukaryotic photosynthetic organisms. Here we found the expression of the chloroplast psb30 gene in the green alga Chlamydomonas reinhardtii by immunoblotting and Psb30 is exclusively co-purifies with PSII core complex and is significantly reduced in PSII-deficient mutants. Partial disintegration of PSII core complex and subsequent fractionation of the resulting subcomplexes revealed that Psb30 is exclusively associated with PSII reaction center. We have generated chloroplast transformants in which the psb30 gene is disrupted and the resulting ΔPsb30 cells showed decreased oxygen evolution activity by 15%, grew photosynthetically under moderate light, and displayed increased sensitivity to high light relative to wild type. We conclude that Psb30 is a PSII reaction center subunit and is required for optimal PSII function under high light environments.     

Response of brassinosteroid-treated oilseed rape cotyledons to infection with the wild type and HR-mutant of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> or with <Emphasis Type="Italic">P. fluorescence</Emphasis>

The aim of the study has been to determine the protection effect of brassinosteroid (BR₂₇) in oilseed rape cotyledons against infection by an incompatible wild type of, a hypersensitive response mutant of and saprophytic Pseudomonas bacteria. In this paper, membrane permeability, PSII efficiency and metabolic activity were analysed. The following strains of Pseudomonans were used: P. syringae pv. syringae (Ps), P. syringae pv. syringae hrcC mutant (Pm) and P. fluorescence (Pf). The study was carried out using two cultivars of spring oilseed rape (Brassica napus L.): ‘Licosmos’ and ‘Huzar’. Pre-treatment of cotyledons with BR₂₇ caused about 50–70% increase in ion leakage for both cultivars. However, BR₂₇ significantly decreased ion leakage from cotyledons inoculated with Ps in both cultivars. Infection with Ps and Pf caused disturbances of energy flow in PSII by lowering its efficiency in rape cotyledons. We noted insignificant impact of 24-epibrassinolide on PSII efficiency if compared to absolute control, but generally it had a positive effect in plants infected with bacteria. The values of heat flow in all treatments, except for cotyledons infected with Ps, decreased during 20 h after inoculation. However, the curves of heat flow for Ps-infected cotyledons showed a completely different pattern with at least two peaks. BR₂₇ pre-treated cotyledons infected with Ps had higher heat flow in comparison to Ps infected ones. BR₂₇ treatment did not change specific enthalpy of cotyledon growth (Δgh) for both cultivars if compared with absolute control. However, infection with Ps markedly increased Δgh values by about 200% for both cultivars. We suggested protective action of BR₂₇ in oilseed rape cotyledons after bacterial infection with Pseudomonas.     

Application of Site‐Directed Lipase Mutants on Regioselective Acylation of Monosaccharides

Mutants F344V and F345V of Candida rugosa lipase1 (CRL1) were tested in acylation reactions of monosaccharide derivatives 1–8, in order to study the regioselectivity, and the substrate specificity of lipase variants towards unnatural substrates, such as carbohydrates. Mutant F344V showed a better reaction kinetics and/or regioselectivity then the wild type enzyme with several substrates while mutant F345V was inefficient in most cases. With the aim of correlating experimental data with the structural features of the enzyme and substrates, the interaction of substrates methyl 4,6‐O‐benzylidene‐α‐d‐glucopyranoside (5a) and 4,6‐O‐benzylidene‐α‐d‐galactopyranoside (6a) with the wild type enzyme and the mutant F344V was investigated, using a molecular modelling approach.