▪ Abstract Plant responses to salinity stress are reviewed with emphasis on molecular mechanisms of signal transduction and on the physiological consequences of altered gene expression that affect biochemical reactions downstream of stress sensing. We make extensive use of comparisons with model organisms, halophytic plants, and yeast, which provide a paradigm for many responses to salinity exhibited by stress-sensitive plants. Among biochemical responses, we emphasize osmolyte biosynthesis and function, water flux control, and membrane transport of ions for maintenance and re-establishment of homeostasis. The advances in understanding the effectiveness of stress responses, and distinctions between pathology and adaptive advantage, are increasingly based on transgenic plant and mutant analyses, in particular the analysis of Arabidopsis mutants defective in elements of stress signal transduction pathways. We summarize evidence for plant stress signaling systems, some of which have components analogous to t...

Plant cellular and molecular responses to high salinity.

The amino acid sequences of 301 glycosyl hydrolases and related enzymes have been compared. A total of 291 sequences corresponding to 39 EC entries could be classified into 35 families. Only ten sequences (less than 5% of the sample) could not be assigned to any family. With the sequences available for this analysis, 18 families were found to be monospecific (containing only one EC number) and 17 were found to be polyspecific (containing at least two EC numbers). Implications on the folding characteristics and mechanism of action of these enzymes and on the evolution of carbohydrate metabolism are discussed. With the steady increase in sequence and structural data, it is suggested that the enzyme classification system should perhaps be revised.

A classification of glycosyl hydrolases based on amino acid sequence similarities.

Copper is an essential trace element in living systems, present in the parts per million concentration range. It is a key cofactor in a diverse array of biological oxidation-reduction reactions. These involve either outer-sphere electron transfer, as in the blue copper proteins and the Cu{sub A} site of cytochrome oxidase and nitrous oxide redutase, or inner-sphere electron transfer in the binding, activation, and reduction of dioxygen, superoxide, nitrite, and nitrous oxide. Copper sites have historically been divided into three classes based on their spectroscopic features, which reflect the geometric and electronic structure of the active site: type 1 (T1) or blue copper, type 2 (T2) or normal copper, and type 3 (T3) or coupled binuclear copper centers. 428 refs.

Multicopper Oxidases and Oxygenases

Lignins are complex natural polymers resulting from oxidative coupling of, primarily, 4-hydroxyphenylpropanoids. An understanding of their nature is evolving as a result of detailed structural studies, recently aided by the availability of lignin-biosynthetic-pathway mutants and transgenics. The currently accepted theory is that the lignin polymer is formed by combinatorial-like phenolic coupling reactions, via radicals generated by peroxidase-H2O2, under simple chemical control where monolignols react endwise with the growing polymer. As a result, the actual structure of the lignin macromolecule is not absolutely defined or determined. The ``randomness'' of linkage generation (which is not truly statistically random but governed, as is any chemical reaction, by the supply of reactants, the matrix, etc.) and the astronomical number of possible isomers of even a simple polymer structure, suggest a low probability of two lignin macromolecules being identical. A recent challenge to the currently accepted theory of chemically controlled lignification, attempting to bring lignin into line with more organized biopolymers such as proteins, is logically inconsistent with the most basic details of lignin structure. Lignins may derive in part from monomers and conjugates other than the three primary monolignols (p-coumaryl, coniferyl, and sinapyl alcohols). The plasticity of the combinatorial polymerization reactions allows monomer substitution and significant variations in final structure which, in many cases, the plant appears to tolerate. As such, lignification is seen as a marvelously evolved process allowing plants considerable flexibility in dealing with various environmental stresses, and conferring on them a striking ability to remain viable even when humans or nature alter ``required'' lignin-biosynthetic-pathway genes/enzymes. The malleability offers significant opportunities to engineer the structures of lignins beyond the limits explored to date.

/pdf/lignins-natural-polymers-from-oxidative-coupling-of-4-oxcvb84q0y.pdf

Lignins: Natural polymers from oxidative coupling of 4-hydroxyphenyl- propanoids

Hemicelluloses for fuel ethanol: A review.

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic beta-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with L-glutamate. This enzyme required pyridoxal 5'-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25 degreesC and had Kms of 9.1 mM for L-glutamate and 4.5 mM for DL-ASA. DABA acetyltransferase catalyzed acetylation of DABA to gamma-N-acetyl-alpha,gamma-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20 degreesC in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15 degreesC in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0. 77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30 degreesC.

Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

cDNA clones for ascorbate oxidase were isolated from a cDNA library made from cucumber (Cucumis sativus) fruit mRNA. The library was screened with synthetic oligonucleotides that encode the NH2-terminal sequence of this enzyme. Nucleotide sequence analysis of the cloned cDNA inserts revealed a 1761-base-pair open reading frame that encoded an NH2-terminal signal peptide of 33 amino acids and a mature enzyme of 554 amino acids (Mr, 62,258). The amino acid sequence deduced from nucleotide sequence analysis agrees with the NH2-terminal amino acid sequence of the purified ascorbate oxidase, as determined by microsequencing methods. Cucumber ascorbate oxidase contained four histidine-rich regions with striking sequence homology to the corresponding parts of the other multicopper oxidases such as Neurospora crassa laccase and human ceruloplasmin and, to some extent, to a low molecular weight copper protein such as plastocyanin. Moreover, these data further support the hypothesis that the small blue copper proteins and the multicopper oxidases have evolved from the same ancestral gene. By RNA blot hybridization analysis, the mRNA for the ascorbate oxidase was found to be abundant in cucumber fruit tissue while expressed at very low levels in leaf and root tissues.

https://www.pnas.org/content/pnas/86/4/1239.full.pdf

Primary structure of cucumber (Cucumis sativus) ascorbate oxidase deduced from cDNA sequence: homology with blue copper proteins and tissue-specific expression.

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19–23 (1976)] with additional sequences at the N and C terminal. All three inserts included in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91–94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S–S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5′ upstream and a poly(A) signal 3′ downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1988.tb14052.x

Structure of the horseradish peroxidase isozyme C genes.

Bacillus amyloliquefaciens 321S cells were immobilized with 3.4% κ-carrageenan gel in bead form, and α-amylase production by the immobilized cells was studied. Cells in the gel, after the population reached maximum were restricted to a layer of 50 μm thickness, from the surface of the gel, suggesting that oxygen diffusion is the growth limiting factor. The specific respiratory activity and the growth rate of the entrapped cells under such conditions were 1/2 and 1/5 ∼1/10, respectively, that of free cells. In spite of the repressed respiration and growth, the specific rate of α-amylase production of the entrapped cells reached the maximum value of free cells or higher. In continuous culture, in an aerated vessel with a volume ratio of gel beads to medium of 1:2, the maximum production rate of α-amylase was obtained at a dilution rate of 1.0 h−1, which was double the maximum specific growth rate of the strain. These results showed that bacterial α-amylase production, which is a nongrowth-associated type of synthesis was achieved with the use of immobilized cells.

Physiology of α-amylase production by immobilized Bacillus amyloliquefaciens

Extracellular endoxylanase (l,4-β-d-xylan xylanohydrolase [EC 3.2.1.8]) produced by Bacillus pumilus IPO, an isolate from soil in Thailand, was purified to homogeneity. The optimum pH and temperature of the purified xylanase for hydrolysis of larchwood xylan were 6.5 and 40°C. Its molecular weight was estimated to be 24,000 dalton by SDS-polyacrylamide gel electrophoresis and 20,000 dalton by equilibrium centrifugation. The number of amino acid residues per molecule was calculated to be 185, including one cysteine.The maximum degree of hydrolysis of larchwood xylan by the purified xylanase was 25%; xylobiose, xylotriose, xylotetraose and xylopentaose but not xylose were detected as end-products. trans-Xylosidase activity was indicated by the formation of xylobiose and xylotetraose from xylotriose and of xylopentaose and hexaose in addition to biose and triose from xylotetraose. Quantification of hydrolysis products indicated that the enzyme′s greatest affinity was for the 2nd to 6th β-xylosidic linkages f...

Atsuhiko Shinmyo

Papers

Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

Primary structure of cucumber (Cucumis sativus) ascorbate oxidase deduced from cDNA sequence: homology with blue copper proteins and tissue-specific expression.

Structure of the horseradish peroxidase isozyme C genes.

Physiology of α-amylase production by immobilized Bacillus amyloliquefaciens

Purification and properties of endoxylanase produced by Bacillus pumilus.