Glutathione improves survival of cryopreserved embryogenic calli of <Emphasis Type="Italic">Agapanthus praecox</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis>

The effect of supplementation of reduced glutathione (GSH) to cryoprotectant solution on the generation of reactive oxygen species (ROS) (e.g., H₂O₂, OH^·, and O ₂ ^·? ) and antioxidants (e.g., SOD, POD, CAT, AsA, and GSH), as well as membrane lipid peroxidation (i.e., MDA content) mitigation in cryopreserving of embryogenic calli (EC) of Agapanthus praecox subsp. orientalis was investigated. The vitrification-based cryopreservation method was used in this study. The addition of GSH at a final concentration of 0.08 mM to the cryoprotectant solution has significantly improved cryotolerance of A. praecox EC. The EC post-thaw survival rate increased by 68.34 % using the cryoprotectant solution containing 0.08 mM GSH as compared to the control (GSH-free). EC treated with GSH displayed the reduction in  OH^· generation activity and the contents of H₂O₂ and MDA, as well as enhancement in the inhibition of O ₂ ^·? generation and the antioxidant activity. Treatment with exogenous GSH also increased endogenous AsA and GSH contents after dehydration step. Expression of stress-responsive genes, e.g., peroxidase (POD), peroxiredoxin, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione peroxidase (GPX), was also increased during cryopreservation processes. The expression of DAD1 (Defender against apoptotic cell death) was elevated, while cell death-related protease SBT was suppressed. These results demonstrated that the addition of GSH to cryoprotectant solution affects the ROS level and could effectively improve survival of A. praecox EC through enhancing antioxidant enzyme activities and decreasing cell death.     

<Emphasis Type="Italic">In vitro</Emphasis> Blastogenesis inhibited by <Emphasis Type="Italic">Erwinia carotovora</Emphasis><Emphasis Type="SmallCaps">L</Emphasis>-Asparaginase

Escherichia coliL-asparaginase, an antileukaemic agent in man¹, inhibits in vitro mitogen or antigen-induced blastogenesis in man^2,3 and in animals (M. Bennett, E. G. Mayhew and T. Han, unpublished data) and suppresses bone-marrow derived antibody precursor cells in the mouse⁴. We now report that another L-asparaginase preparation—from Erwinia carotovora—also possesses antileukaemic activity^5,6 and has a more pronounced immunosuppressive effect on in vitro blastogenesis than the E. coli enzyme.     

Effect of calcium ions on electron transfer between hemes <Emphasis Type="Italic">a</Emphasis> and <Emphasis Type="Italic">a</Emphasis><Subscript>3</Subscript> in cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

Kinetics of the reduction of the hemes in cytochrome c oxidase in the presence of high concentration of ruthenium(III)hexaammine chloride was examined using a stopped-flow spectrophotometer. Upon mixing of the oxidized enzyme with dithionite and Ru(NH₃) ₆ ³⁺ , three well-resolved phases were observed: heme a reduction reaching completion within a few milliseconds is followed by two slow phases of heme a ₃ reduction. The difference spectrum of heme a ₃ reduction in the visible region is characterized by a maximum at ～612 nm, rather than at 603 nm as was believed earlier. It is shown that in the case of bovine heart cytochrome c oxidase containing a special cation-binding site in which reversible binding of calcium ion occurs, heme a ₃ reduction is slowed down by low concentrations of Ca²⁺. The effect is absent in the case of the bacterial cytochrome oxidase in which the cation-binding site contains a tightly bound Ca²⁺ ion. The data corroborate the inhibition of the cytochrome oxidase enzymatic activity by Ca²⁺ ions discovered earlier and indicate that the cation affects intramolecular electron transfer.     

<Emphasis Type="Italic">C7</Emphasis>-prenylation of tryptophanyl and <Emphasis Type="Italic">O</Emphasis>-prenylation of tyrosyl residues in dipeptides by an <Emphasis Type="Italic">Aspergillus terreus</Emphasis> prenyltransferase

During our search for novel prenyltransferases, a putative gene ATEG_04218 from Aspergillus terreus raised our attention and was therefore amplified from strain DSM 1958 and expressed in Escherichia coli. Biochemical investigations with the purified recombinant protein and different aromatic substrates in the presence of dimethylallyl diphosphate revealed the acceptance of all the tested tryptophan-containing cyclic dipeptides. Structure elucidation of the main enzyme products by NMR and MS analyses confirmed the attachment of the prenyl moiety to C-7 of the indole ring, proving the identification of a cyclic dipeptide C7-prenyltransferase (CdpC7PT). For some substrates, reversely C3- or N1-prenylated derivatives were identified as minor products. In comparison to the known tryptophan-containing cyclic dipeptide C7-prenyltransferase CTrpPT from Aspergillus oryzae, CdpC7PT showed a much higher substrate flexibility. It also accepted cyclo-l-Tyr-l-Tyr as substrate and catalyzed an O-prenylation at the tyrosyl residue, providing the first example from the dimethylallyltryptophan synthase (DMATS) superfamily with an O-prenyltransferase activity towards dipeptides. Furthermore, products with both C7-prenyl at tryptophanyl and O-prenyl at tyrosyl residue were detected in the reaction mixture of cyclo-l-Trp-l-Tyr. Determination of the kinetic parameters proved that (S)-benzodiazepinedione consisting of a tryptophanyl and an anthranilyl moiety was accepted as the best substrate with a K _M value of 204.1 μM and a turnover number of 0.125 s^?1. Cyclo-l-Tyr-l-Tyr was accepted with a K _M value of 1,411.3 μM and a turnover number of 0.012 s^?1.     

Decrease of phosphate concentration in the medium by <Emphasis Type="Italic">Brevibacterium casei</Emphasis> cells

Brevibacteria able to decrease phosphate concentration in the medium are of interest for the study of the role of bacteria in the phosphorus cycle and for development of biotechnology of phosphate removal from waste. Brevibacterium casei, Brevibacterium linens, and Brevibacterium epidermidis grown in media with initial phosphorus concentrations of 1–11 mM were shown to decrease its concentration by 90%. The composition of the incubation medium required for B. casei to carry out this process was established. This process occurs in the absence of glucose but requires the presence of Mg²⁺, NH ₄ ⁺ , and α-ketoglutarate. The latter two components may be replaced by amino acids metabolized to NH ₄ ⁺ and α-ketoglutarate: histidine, arginine, glutamine, proline, or glutamic acid. No formation of insoluble phosphate salts was observed when the media were incubated under the same conditions with heat-inactivated cells or without cells at pH 7–8.5.     

<Emphasis Type="Italic">Methylophilaceae</Emphasis> and <Emphasis Type="Italic">Hyphomicrobium</Emphasis> as target taxonomic groups in monitoring the function of methanol-fed denitrification biofilters in municipal wastewater treatment plants

Molecular monitoring of bacterial communities can explain and predict the stability of bioprocesses in varying physicochemical conditions. To study methanol-fed denitrification biofilters of municipal wastewater treatment plants, bacterial communities of two full-scale biofilters were compared through fingerprinting and sequencing of the 16S rRNA genes. Additionally, 16S rRNA gene fingerprinting was used for 10-week temporal monitoring of the bacterial community in one of the biofilters. Combining the data with previous study results, the family Methylophilaceae and genus Hyphomicrobium were determined as suitable target groups for monitoring. An increase in the relative abundance of Hyphomicrobium-related biomarkers occurred simultaneously with increases in water flow, NO _x ^? load, and methanol addition, as well as a higher denitrification rate, although the dominating biomarkers linked to Methylophilaceae showed an opposite pattern. The results indicate that during increased loading, stability of the bioprocess is maintained by selection of more efficient denitrifier populations, and this progress can be analyzed using simple molecular fingerprinting.     

Oxidative processes at initial stages of interaction of nodule bacteria (<Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis>) and pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.): A review

A possible physiological mechanism of legume-Rhizobium symbiosis, consisting in regulation of the intensity of oxidative processes by the macrosymbiont in response to infection with Rhizobium, was analyzed using our own and published data. The results used in the analysis included data on the content of reactive oxygen species (O ₂ ^·? and H₂O₂), activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase), and intensity of lipid peroxidation proceeding with the involvement of lipophilic phenolic compounds of the microsymbiont.     

Biocidal effect of (<Emphasis Type="Italic">E</Emphasis>)-anethole on the cyanobacterium <Emphasis Type="Italic">Aphanizomenon gracile</Emphasis> Lemmermann

Biocidal natural substances of botanical origin offer a promising ecofriendly option for controlling toxic cyanobacteria. Herein, we study 11 essential oils and some of their major components for their activity on Aphanizomenon gracile. On the basis of our results we support that Origanum vulgare and O. dictamnus, Ocimum basilicum, Eucalyptus meliodora, Melissa officinalis, and Pimpinella anisum exhibited the strongest activities, and the IC_50/1d values of the extracts were calculated to be between 168.43 and 241.97 μg mL^?1. When the major components of the biocidal essential oils were tested individually, (E)-anethole was found active, exhibiting an IC_50/1d value of 71.35 μg mL^?1. On the other hand, the half-life (t _1/2 ) of (E)-anethole was calculated at 1 h. A preliminary attempt of (E)-anethole microencapsulation was conducted, in order to slowly release this biocidal agent, increasing the residual life under open air conditions and thus the biological activity. Results were promising since the microencapsulated product exhibited better activity than did the non-formulated (E)-anethole. This is a first report on the biocidal activity of EOs and (E)-anethole on A. gracile and a preliminary indication of the microencapsulated (E)-anethole potential use as a natural biocidal in fresh waters.

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Graphical abstract Filaments of Aphanizomenon gracile capable of bloom forming (strain SAG 31.79, in a batch culture) and (E)-anethole

     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

Combinatorial analysis of enzymatic bottlenecks of <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine pathway by <Emphasis Type="Italic">p</Emphasis>-coumaric acid production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Objective

To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives.

Result

When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l^?1, respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l^?1) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain.

Conclusion

Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.

     

Identification of <Emphasis Type="Italic">Pm58</Emphasis> from <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

Key message

A novel powdery mildew-resistance gene, designated Pm58, was introgressed directly from Aegilops tauschii to hexaploid wheat, mapped to chromosome 2DS, and confirmed to be effective under field conditions. Selectable KASP? markers were developed for MAS.

Abstract

Powdery mildew caused by Blumeria graminis (DC.) f. sp. tritici (Bgt) remains a significant threat to wheat (Triticum aestivum L.) production. The rapid breakdown of race-specific resistance to Bgt reinforces the need to identify novel sources of resistance. The d-genome species, Aegilops tauschii, is an excellent source of disease resistance that is transferrable to T. aestivum. The powdery mildew-resistant Ae. tauschii accession TA1662 (2n?=?2x?=?DD) was crossed directly with the susceptible hard white wheat line KS05HW14 (2n?=?6x?=?AABBDD) followed by backcrossing to develop a population of 96 BC₂F₄ introgression lines (ILs). Genotyping-by-sequencing was used to develop a genome-wide genetic map that was anchored to the Ae. tauschii reference genome. A detached-leaf Bgt assay was used to screen BC₂F_4:6 ILs, and resistance was found to segregate as a single locus (χ?=?2.0, P value?=?0.157). The resistance gene, referred to as Pm58, mapped to chromosome 2DS. Pm58 was evaluated under field conditions in replicated trials in 2015 and 2016. In both years, a single QTL spanning the Pm58 locus was identified that reduced powdery mildew severity and explained 21% of field variation (P value?<?0.01). KASP? assays were developed from closely linked GBS-SNP markers, a refined genetic map was developed, and four markers that cosegregate with Pm58 were identified. This novel source of powdery mildew-resistance and closely linked genetic markers will support efforts to develop wheat varieties with powdery mildew resistance.

     

New insights into the photochemistry of carotenoid spheroidenone in light-harvesting complex 2 from the purple bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Light-harvesting complex 2 (LH2) from the semi-aerobically grown purple phototrophic bacterium Rhodobacter sphaeroides was studied using optical (static and time-resolved) and resonance Raman spectroscopies. This antenna complex comprises bacteriochlorophyll (BChl) a and the carotenoid spheroidenone, a ketolated derivative of spheroidene. The results indicate that the spheroidenone-LH2 complex contains two spectral forms of the carotenoid: (1) a minor, “blue” form with an S₂ (1¹B _u ⁺ ) spectral origin band at 522 nm, shifted from the position in organic media simply by the high polarizability of the binding site, and (2) the major, “red” form with the origin band at 562 nm that is associated with a pool of pigments that more strongly interact with protein residues, most likely via hydrogen bonding. Application of targeted modeling of excited-state decay pathways after carotenoid excitation suggests that the high (92%) carotenoid-to-BChl energy transfer efficiency in this LH2 system, relative to LH2 complexes binding carotenoids with comparable double-bond conjugation lengths, derives mainly from resonance energy transfer from spheroidenone S₂ (1¹B _u ⁺ ) state to BChl a via the Q_x state of the latter, accounting for 60% of the total transfer. The elevated S₂ (1¹B _u ⁺ ) → Q_x transfer efficiency is apparently associated with substantially decreased energy gap (increased spectral overlap) between the virtual S₂ (1¹B _u ⁺ ) → S₀ (1¹A _g ^? ) carotenoid emission and Q_x absorption of BChl a. This reduced energetic gap is the ultimate consequence of strong carotenoid–protein interactions, including the inferred hydrogen bonding.     

<Emphasis Type="Italic">Zunongwangia flava</Emphasis> sp. nov., belonging to the family <Emphasis Type="Italic">Flavobacteriaceae</Emphasis>, isolated from <Emphasis Type="Italic">Salicornia europaea</Emphasis>

A yellow pigmented bacterium designated strain MBLN094^T within the family Flavobacteriaceae was isolated from a halophyte Salicornia europaea on the coast of the Yellow Sea. This strain was a Gram-stain negative, aerobic, non-spore forming, rod-shaped bacterium. Phylogenetic analysis of the 16S rRNA gene sequence of strain MBLN094^T was found to be related to the genus Zunongwangia, exhibiting 16S rRNA gene sequence similarity values of 97.0, 96.8, 96.4, and 96.3% to Zunongwangia mangrovi P2E16^T, Z. profunda SM-A87^T, Z. atlantica 22II14-10F7^T, and Z. endophytica CPA58^T, respectively. Strain MBLN094^T grew at 20?37°C (optimum, 25?30°C), at pH 6.0?10.0 (optimum, 7.0?8.0), and with 0.5?15.0% (w/v) NaCl (optimum, 2.0?5.0%). Menaquinone MK-6 was the sole respiratory quinone. The polar lipids were phosphatidylethanolamine, two unidentified aminolipids, and four unidentified lipids. Major fatty acids were iso-C_17:0 3-OH, summed feature 3 (C_16:1ω6c and/or C16:1 ω7c), and iso-C_15:0. The genomic DNA G + C content was 37.4 mol%. Based on these polyphasic taxonomic data, strain MBLN094^T is considered to represent a novel species of the genus Zunongwangia, for which the name Zunongwangia flava sp. nov. is proposed. The type strain is MBLN094^T (= KCTC 62279^T = JCM 32262^T).     

<Emphasis Type="Italic">Nibribacter flagellatus</Emphasis> sp. nov., isolated from rhizosphere of <Emphasis Type="Italic">Hibiscus syriacus</Emphasis> and emended description of the genus <Emphasis Type="Italic">Nibribacter</Emphasis>

A Gram-stain negative, aerobic, motile by flagella, rod-shaped strain (THG-T16^T) was isolated from rhizosphere of Hibiscus syriacus. Growth occurred at 10–40 °C (optimum 28–30 °C), at pH 6.0–8.0 (optimum 7.0) and at 0–1.0% NaCl (optimum 0%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-T16^T were identified as Nibribacter koreensis KACC 16450^T (98.6%), Rufibacter roseus KCTC 42217^T (94.7%), Rufibacter immobilis CCTCC AB 2013351^T (94.5%) and Rufibacter tibetensis CCTCC AB 208084^T (94.4%). The DNA G+C content of strain THG-T16^T was determined to be 46.7 mol%. DNA–DNA hybridization values between strain THG-T16^T and N. koreensis KACC 16450^T, R. roseus KCTC 42217^T, R. immobilis CCTCC AB 2013351^T, R.tibetensis CCTCC AB 208084^T were 33.5?±?0.5% (31.7?±?0.7% reciprocal analysis), 28.1?±?0.2% (25.2?±?0.2%), 17.1?±?0.9% (10.2?±?0.6%) and 8.1?±?0.3% (5.2?±?0.1%). The polar lipids were identified as phosphatidylethanolamine, two unidentified aminophospholipids, an unidentified aminolipid and three unidentified lipids. The quinone was identified as MK-7 and the polyamine as sym-homospermidine. The major fatty acids were identified as C_16:1 ω5c, C_17:1 ω6c, iso-C_15:0, summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c) and summed feature 4 (iso-C_17:1 I and/or anteiso-C_17:1 B). On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics, and DNA–DNA hybridization data, strain THG-T16^T represents a novel species of the genus Nibribacter, for which the name Nibribacter flagellatus sp. nov. is proposed. The type strain is THG-T16^T(=?KACC 19188^T?=?CCTCC AB 2016246^T).     

Karyo-taxonomic study of the genus<Emphasis Type="Italic">Pseudolysimachion</Emphasis> (<Emphasis Type="Italic">Scrophulariaceae</Emphasis>) in the Czech Republic and Slovakia

Flow cytometry was used to determine ploidy levels in the Czech and Slovak taxa of the genusPseudolysimachion (W.D.J. Koch)Opiz (=Veronica auct. p.p.,Scrophulariaceae). In total, 123 populations from the Czech Republic, Slovakia, Ukraine (one locality), Austria (one locality) and Hungary (one locality) were analyzed. InP. maritimum (L.)Á. Löve etD. Löve andP. spicatum (L.)Opiz, two cytotypes were found: diploid (2n=2x=34) and tetraploid (2n=4x=68). In both species the tetraploid cytotype predominated (P. maritimum: 41 tetraploid populations out of 45;P. spicatum: 57 tetraploid populations out of 58). The two cytotypes ofP. maritimum have no taxonomic significance because ploidy level is not obviously correlated with morphology, distribution pattern or ecology. Tetraploid populations ofP. spicatum belong to two morphologically different subspecies, subsp.spicatum and subsp.fischeri Trávní?ek. The diploid cytotype (one population only) should be provisionally classified as a third subspecies ofP. spicatum, which is morphologically similar to the Asian subsp.porphyrianum (Pavlov)Trávní?ek. Only diploid plants (2n=2x=34) ofP. orchideum (Crantz)Wraber were found; all 13 populations that were analyzed belong toP. orchideum s.str. One diploid population sample ofP. spurium subsp.foliosum (Waldst. etKit.)Holub (2n=2x=34) and one tetraploid sample ofP. incanum subsp.pallens (Host)Trávní?ek (2n=4x=68) were also analyzed. In addition, three tetraploid populations of hybrid origin were investigated:P. maritimum ×P. spicatum subsp.spicatum (one population) andP. maritimum ×P. spurium subsp.foliosum (two populations). While hybrid plants ofP. maritimum ×P. spicatum arose from tetraploid parental species, plants ofP. maritimum ×P. spurium probably resulted from a cross between tetraploidP. maritimum and diploidP. spurium. The putative origin and evolutionary importance of polyploids in thePseudolysimachion are discussed.     

Synthesis of high-molecular-mass polyhydroxybutyrate from methanol in <Emphasis Type="Italic">Methyloligella halotolerans</Emphasis> C2

The influence of the concentrations of carbon and energy sources (methanol) and nitrogen (NH ₄ ⁺ ) on the yield and molecular mass (Mm) of poly-β-hydroxybutyrate (PHB) synthesized by the novel methylotroph Methyloligella halotolerans C2 was investigated. It was shown that the maximum concentrations of NH ₄ ⁺ and methanol in the medium for PHB biosynthesis were 0.15 g/L and 1 ± 0.2 mL/L, respectively. A unique high-molecular (8000–10000 kDa) PHB was obtained with NH ₄ ⁺ and methanol concentrations of 0.12 g/L and 1.25 mL/L, respectively, in a fermenter. The main physicochemical and mechanical properties of the obtained bioplastic with a molecular mass of 10000 kDa were determined.     

Nitrogen uptake and allocation in <Emphasis Type="Italic">Populus simonii</Emphasis> in different seasons supplied with isotopically labeled ammonium or nitrate

Key message

The total uptake of ¹⁵ NO ₃ -N was twofold higher than that of ¹⁵ NH ₄ -N when supplied with ammonium and/or nitrate in different seasons; the seedlings fertilized with NO ₃ -N had good growth with high photosynthetic rate and total biomass.

Abstract

Appropriate fertilization is crucial for maximum plant growth and improving nitrogen use efficiency. Poplar is an important fast-growing tree species for biomass production, however, little is known about fertilizer management of poplar plantations growing on barren soil in different seasons. To understand nitrogen uptake and allocation of Populus simonii supplied with different forms of nitrogen in different seasons, we determined nitrogen uptake and allocation of P. simonii potted seedlings after a 4-day supply of ¹⁵NH₄-N, ¹⁵NO₃-N, ¹⁵NH₄NO₃, and NH ₄ ¹⁵ NO₃ in May, July, and September. The total ¹⁵N uptake was twofold higher when supplied with sole ¹⁵NO₃-N compared to sole ¹⁵NH₄-N in all the investigated seasons. In the presence of ammonium nitrate (¹⁵NH₄NO₃ and NH ₄ ¹⁵ NO₃), the total ¹⁵N uptake was two times higher when supplied with NH ₄ ¹⁵ NO₃ compared to ¹⁵NH₄NO₃. Per unit biomass, the ¹⁵N-uptake ability of fine roots was higher in May and July compared to that in September. ¹⁵N was present mainly in leaves in May and July, and was mainly stored in roots and stems in autumn. The effect of nitrogen on the growth of P. simonii seedlings was studied by fertilizing with NH₄-N, NO₃-N, and NH₄NO₃ for 8 weeks. The seedlings fertilized with NO₃-N had good growth with high photosynthetic rate and total biomass indicating that NO₃-N is crucial for P. simonii growth. These data contribute to understand the nitrogen uptake in different seasons in trees supplied with different forms of nitrogen. This provides important theoretical bases for fertilizer management of poplar plantations.

     

Characterization of aspartate kinase and homoserine dehydrogenase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> IWJ001 and systematic investigation of <Emphasis Type="SmallCaps">l</Emphasis>-isoleucine biosynthesis

Previously we have characterized a threonine dehydratase mutant TD^F383V (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS^{P176S, D426E, L575W} (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best l-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK^A279T (encoded by lysC1) and a homoserine dehydrogenase mutant HD^G378S (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK^A279T is completely resistant to feed-back inhibition by l-threonine and l-lysine, and that HD^G378S is partially resistant to l-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive l-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from l-lysine (decreased by 50.1 %) to l-threonine (4.85 g/L) with minor l-isoleucine and no l-homoserine accumulation, further co-expressing ilvA1 completely depleted l-threonine and strongly shifted carbon flux from l-lysine (decreased by 83.0 %) to l-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD^F383V might be the main driving force for l-isoleucine over-synthesis in this case, and the partially feed-back resistant HD^G378S might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering.     

Identification of 21 novel <Emphasis Type="Italic">S-RNase</Emphasis> alleles and determination of <Emphasis Type="Italic">S</Emphasis>-genotypes in 66 loquat (<Emphasis Type="Italic">Eriobotrya</Emphasis>) accessions

As observed in other self-incompatible species in the Pyrinae subtribe, loquat (Eriobotrya japonica) demonstrates gametophytic self-incompatibility that is controlled by the S-locus, which encodes a polymorphic stylar ribonuclease (S-RNase). This allows the female reproductive organ (style) to recognize and reject the pollen from individuals with the same S-alleles, but allows the pollen from individuals with different S-alleles to effect fertilization. The S-genotype is therefore an important consideration in breeding strategies and orchard management. In an attempt to optimize the selection of parental lines in loquat production, the S-RNase alleles of 35 loquat cultivars and their 26 progeny, as well as five wild loquat species, were identified and characterized in this study. The best pollinizer cultivar combinations were also explored. A total of 28 S-alleles were detected, 21 of which constituted novel S-RNase alleles. The S-haplotypes S₂ and S₆ were the most frequent, followed by S ₂₉ , S ₃₁ , S ₅ , S ₂₄ , S ₂₈ , S ₃₃ , S ₃₄ , S ₃₂ , and S ₁₅ , while the rare alleles S ₁ , S ₉ , S ₁₄ , S ₁₆ , S ₁₇ , S ₁₈ , S ₁₉ , S ₂₀ , S ₂₁ , S ₂₂ , S ₂₃ , S ₂₇ , and S ₃₅ were only observed in one of the accessions tested. Moreover, the S-genotypes of five wild loquat species (E. prinoides, E. bengalensis, E. prinoides var. dadunensis, E. deflexa, and E. japonica) are reported here for the first time. The results will not only facilitate the selection of suitable pollinators for optimal orchard management, but could also encourage the crossbreeding of wild loquat species to enhance the genetic diversity of loquat cultivars.     

Statistical techniques for modeling of <Emphasis Type="Italic">Corylus</Emphasis>, <Emphasis Type="Italic">Alnus</Emphasis>, and <Emphasis Type="Italic">Betula</Emphasis> pollen concentration in the air

Prediction of allergic pollen concentration is one of the most important goals of aerobiology. Past studies have used a broad range of modeling techniques; however, the results cannot be directly compared owing to the use of different datasets, validation methods, and evaluation metrics. The main aim of this study was to compare nine statistical modeling techniques using the same dataset. An additional goal was to assess the importance of predictors for the best model. Aerobiological data for Corylus, Alnus, and Betula pollen counts were obtained from nine cities in Poland and covered between five and 16 years of measurements. Meteorological data from the AGRI4CAST project were used as a predictor variables. The results of 243 final models (3 taxa \(\times\)  9 cities \(\times\) 9 techniques) were validated using a repeated k-fold cross-validation and compared using relative and absolute performance statistics. Afterward, the variable importance of predictors in the best models was calculated and compared. Simple models performed poorly. On the other hand, regression trees and rule-based models proved to be the most accurate for all of the taxa. Cumulative growing degree days proved to be the single most important predictor variable in the random forest models of Corylus, Alnus, and Betula. Finally, the study suggested potential improvements in aerobiological modeling, such as the application of robust cross-validation techniques and the use of gridded variables.