Analysis of rice glycosyl hydrolase family 1 and expression of Os4bglu12 β-glucosidase

Background

Glycosyl hydrolase family 1 (GH1) β-glucosidases have been implicated in physiologically important processes in plants, such as response to biotic and abiotic stresses, defense against herbivores, activation of phytohormones, lignification, and cell wall remodeling. Plant GH1 β-glucosidases are encoded by a multigene family, so we predicted the structures of the genes and the properties of their protein products, and characterized their phylogenetic relationship to other plant GH1 members, their expression and the activity of one of them, to begin to decipher their roles in rice.

Results

Forty GH1 genes could be identified in rice databases, including 2 possible endophyte genes, 2 likely pseudogenes, 2 gene fragments, and 34 apparently competent rice glycosidase genes. Phylogenetic analysis revealed that GH1 members with closely related sequences have similar gene structures and are often clustered together on the same chromosome. Most of the genes appear to have been derived from duplications that occurred after the divergence of rice and Arabidopsis thaliana lineages from their common ancestor, and the two plants share only 8 common gene lineages. At least 31 GH1 genes are expressed in a range of organs and stages of rice, based on the cDNA and EST sequences in public databases. The cDNA of the Os4bglu12 gene, which encodes a protein identical at 40 of 44 amino acid residues with the N-terminal sequence of a cell wall-bound enzyme previously purified from germinating rice, was isolated by RT-PCR from rice seedlings. A thioredoxin-Os4bglu12 fusion protein expressed in Escherichia coli efficiently hydrolyzed β-(1,4)-linked oligosaccharides of 3–6 glucose residues and laminaribiose.

Conclusion

Careful analysis of the database sequences produced more reliable rice GH1 gene structure and protein product predictions. Since most of these genes diverged after the divergence of the ancestors of rice and Arabidopsis thaliana, only a few of their functions could be implied from those of GH1 enzymes from Arabidopsis and other dicots. This implies that analysis of GH1 enzymes in monocots is necessary to understand their function in the major grain crops. To begin this analysis, Os4bglu12 β-glucosidase was characterized and found to have high exoglucanase activity, consistent with a role in cell wall metabolism.     

Determination of β-glucosidase aggregating factor (BGAF) binding and polymerization regions on the maize β-glucosidase isozyme Glu1

β-Glucosidases (Glu1 and Glu2) in maize specifically interact with a lectin called β-glucosidase aggregating factor (BGAF). We have shown that the N-terminal (Glu⁵⁰–Val¹⁴⁵) and the C-terminal (Phe⁴⁶⁶–Ala⁵¹²) regions of maize Glu1 are involved in binding to BGAF. Sequence comparison between sorghum β-glucosidases (dhurrinases, which do not bind to BGAF) and maize β-glucosidases, and the 3D-structure of Glu1 suggested that the BGAF-binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF-binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile⁷²–Thr⁸²) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser¹–Thr²⁹, together with C-terminal region Phe⁴⁶⁶–Ala⁵¹², affects the size of Glu1–BGAF complexes. The dissociation constants (K_d) of chimeric β-glucosidase–BGAF interactions also demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile⁷²–Thr⁸² on Glu1 for BGAF binding, we constructed a chimeric sorghum β-glucosidase, Dhr2 (C-11, Dhr2 whose Val⁷²–Glu⁸² region was replaced with the Ile⁷²–Thr⁸² region of Glu1). C-11 binds to BGAF, indicating that the Ile⁷²–Thr⁸² region is indeed a major interaction site on Glu1 involved in BGAF binding.     

ZEIN DEGRADATION IN THE ENDOSPERM OF MAIZE SEEDS DURING GERMINATION

The pattern and sequence of zein degradation in the endosperm of germinating maize seeds were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by immunostaining Western blots with a monoclonal antibody to α-zein and polyclonal antibodies to β-, γ-, and δ-zeins. The results indicated: 1) the degradation of the predominant α-zein fraction (23.8 and 26.7 kD) started on the 5th day after germination (DAG) and continued gradually until the 10th DAG with a small amount remaining undegraded up to the 26th DAG; 2) β-zein (17 kD) began to be degraded on the 2d DAG, and the degradation of the 17 kD polypeptide was completed by the 7th DAG; 3) γ-zeins (27 and 18 kD) were the first zeins to be degraded, and the degradations of γ-zeinl (27 kD) and γ-zein2 (18 kD) were complete by the 3rd and 4th DAG, respectively; and 4) the degradation of δ-zein (10 kD) began on the 4th DAG and was complete by the 7th DAG. Based on these results, the following arrangement of zein polypeptides within the protein bodies is postulated, assuming that the proteolytic events start at the periphery and processed towards the core of the protein body: 1) γ-zeins are situated around the periphery of the protein bodies and are possibly a structural component of the protein body membrane or directly anchored in the membrane; 2) β-zein would be internal to γ-zeins; and 3) α-zein and δ-zein would be in the protein body core. This arrangement is largely consistent with published data on the immunocytochemical localization of zeins, and it indicates that the different classes of zein are not randomly organized within the matrix of the protein body.     

AMYLASE POLYMORPHISM IN CITRUS AND SOME RELATED GENERA

Amylase polymorphism was studied by electrophoresing crude protein extracts from mature leaves of Citrus and some related genera in polyacrylamide gel slabs. A total of 25 isoenzymes distributed within 2 distinct zones were resolved. Cultivars of C. medica were the least polymorphic with 5 isoenzymes, those of C. paradisi were the most polymorphic with 14 isoenzymes; the other taxa were between these two extremes. Cultivars within species, in general, had no qualitative differences in their isoenzymes profiles with the exception of C. aurantifolia, C. limon, and C. aurantium. Certain isoenzymes showed quantitative differences among taxa. Data provided evidence substantiating the presumed hybrid origin of some taxa. The potential of isoenzyme profiles in taxonomic and genetic studies was discussed.     

Investigating the effects of subthalamic Nucleus–Deep brain stimulation on the voice quality

Abstract

Introduction: Deep brain stimulation (DBS) is a standard surgical treatment method which is generally applied to subthalamic nucleus in Parkinson’s patients in cases where medical treatment is insufficient in treating the motor symptoms. It is known that Subthalamic Nucleus Deep Brain Stimulation (STN-DBS) treats many motor symptoms. However, the results of studies on speech and voice vary. The aim of the study is analysing the effect of STN-DBS on the characteristics of voice.

Materials/methods: A total of 12 patients, (8 male–4 female) with an age average of 58.8?±?9.6, who have been applied DBS surgery on STN included in the study. The voice recordings of the patients have been done prior to surgery and 6?months after the surgery. The evaluation of voice has been carried out through the instrumental method. The patients’ voice recordings of the /a,e,i/ vowels have been done. The obtained recordings were evaluated by the Praat programme and the effects on jhitter, shimmer, fundamental frequency (F0) and noise harmonic rate (NHR) were analysed.

Results: Numerical values of F0 of all female participants have been decreased for all of the vowels postoperatively. In the females; jhitter and fraction parameters were found to be significantly different (0.056 and 0.017, perspectively) for the vowel /e/. In addition, p values in the shimmer for vowels /e,i/ were thought to be clinically significant (.087, .079 and .076) respectively. All these changes in second measurements were found to indicate worsening vocal quality after the DBS in females. In males, there is not any significant difference observed between two measures in any of the parameters of any vowels.

Conclusions: Acoustic voice quality deteriorated after STN-DBS predominantly for females however this deterioration was not prominent audio-perceptually. This finding commented as a result of the fact that that voice quality deviance of the participants was not severe.     

ADVENTIVE EMBRYOGENESIS IN CITRUS AND ITS RELATION TO POLLINATION AND FERTILIZATION

Cytological and histological studies of seeds from three facultative apomictic Citrus cultivars show that adventive embryos develop, as a rule, from the first few cell layers of the nucellus adjacent to the embryo sac in the micropylar half and occasionally from the chalazal end. The adventive embryos initiated in nucellar tissue away from the embryo sac and most of those initiated from the chalazal end of the nucellus do not develop beyond the one-celled stage. When two or more embryos are developing in the same seed, the successful development of a given embryo depends on its location in relation to access to nutrients from the endosperm. The presence of a zygote and triploid endosperm in seeds with adventive embryos, the abortion of seed when endosperm degenerates, and the lack of seed set without pollination indicate that pollination and fertilization are essential for in vivo adventive embryogenesis.     

A 23.8-kD alpha-zein with N-terminal sequence and immunological properties similar to 26.7-kD alpha-zeins

A 23.8-kD alpha-zein polypeptide, K55PC7, has been shown to be a truncated member of the 26.7-kD alpha-zein class based on its amino acid composition, N-terminal sequence, and immunological properties. This unusual polypeptide was isolated by chromatographing whole alpha-zein from inbred K55. The N-terminal sequence of K55PC7 is highly homologous to those of 4 putative 26.7-kD alpha-zeins but shows no homology to those of 10 putative alpha-zeins that belong to the 23.8-kD class. Its higher valine and lower phenylalanine contents also suggest that K55PC7 is a member of the 26.7-kD class. In addition, studies with antibodies raised to peptides corresponding to regions unique to each of the two alpha-zein classes indicate that K55PC7 has immunological similarity to 26.7-kD alpha-zeins. Peptide mapping data suggest that K55PC7 is not the putative product of the truncated 26.7-kD alpha-zein gene zA1 isolated from inbred W64A and described by Spena et al. [26]. It appears that K55PC7 occurs as a major component in inbred K55 and is a truncated version of a 26.7-kD alpha-zein, arisen either by an internal deletion or premature termination due to a nonsense mutation.Abbreviations SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - EIF isoelectric focusing - SP sulfopropyl-Sephadex - PC phosphocellulose - DSS disuccinimidyl suberate - MBC m-maleimido-benzoyl-N-hydroxysuccinimide - 2-ME 2-mercaptoethanol     

Comparative study on the inactivation of MS2 and M13 bacteriophages using energetic femtosecond lasers

Femtosecond (fs) laser irradiation techniques are emerging tools for inactivating viruses that do not involve ionizing radiation. In this work, the inactivation of two bacteriophages representing protective capsids with different geometric constraints, that is, the near‐spherical MS2 (with a diameter of 27 nm) and the filamentous M13 (with a length of 880 nm) is compared using energetic visible and near‐infrared fs laser pulses with various energies, pulse durations, and exposure times. Intriguingly, the results show that inactivation using 400 nm lasers is substantially more efficient for MS2 compared to M13. In contrast, using 800 nm lasers, M13 was slightly more efficiently inactivated. For both viruses, the genome was exposed to a harmful environment upon fs‐laser irradiation. However, in addition to the protection of the genome, the metastable capsids differ in many properties required for stepwise cell entry that may explain their dissimilar behavior after (partial) disassembly. For MS2, the dominant mechanism of fs‐laser inactivation was the aggregation of the viral capsid proteins, whereas aggregation did not affect M13 inactivation, suggesting that the dominant mechanism of M13 inactivation was related to breaking of secondary protein links.