Shunnosuke Abe

Methods for isolation and analysis of polyribosomes.

Publisher Summary This chapter discusses methods for the isolation and analysis of polyribosomes. Polysomes consist of two or more ribosomes traversing a strand of mRNA, translating the nucleotide sequence into the corresponding amino acid sequence. One reason for isolating polysomes is to resolve them on sucrose gradients to determine the extent of ribosome loading. The second main reason is to provide a source of mRNA for in vitro translation or cDNA probing. The major factor influencing the number of ribosomes per mRNA molecule is RNase—an enzyme that is difficult to inhibit, and many polysome isolation protocols are designed solely to prevent its action. RNase activity initially causes the conversion of large polysomes into small ones, and, only at later stages, the monosomes accumulate. Disaggregation can also result from ribosome run-off, which leads to the accumulation of monosomes and only a slight shift from large to smaller polysomes.

Evidence for the existence of cytoskeleton-bound polysomes in plants

When conventional, high ionic strength buffers were used for the isolation of polysomes from pea plants, less than 20% were retained in the detergent-insoluble pellet. Reducing Tris, K+ and Mg++ to 10 mM increased retention to 70%, and when a new, microfilament-stabilizing buffer was used, retention increased to 80%. Conditions which favoured polysome pelleting at lower g forces permitted the retention of actin in the pellet. The data are consistent with the hypothesis that higher plants, like animals, contain cytoskeleton-(actin)-bound polysomes.

METHODS FOR ISOLATION AND ANALYSIS OF THE CYTOSKELETON

Publisher Summary This chapter discusses methods for the isolation and analysis of the cytoskeleton (CSK). The plant CSK consists of microfilaments (MFs) and microtubules (MTs) interchanging dynamically with their corresponding monomeric proteins, actin and tubulin, and perhaps of intermediate filaments. Monomeric actin and tubulin have been isolated from many plant tissues and described. The chapter focuses on the isolation of the CSK itself and deals with solubilized monomers. The first methods for isolating relatively intact CSK in amounts sufficient for biochemical analysis employed protoplasts from carrot suspension cells (Hussey et al ). These pioneering methods suffer from two potential drawbacks: (1) they are suitable only for tissues that yield protoplasts easily and (2) the conditions needed to release protoplasts can themselves cause substantial changes in the CSK and thus lead to artifacts (Tan and Boss). Relatively little is known about actin-binding proteins (ABPs) and tubulin-binding proteins (TBPs) and even less about MF-associated proteins (MFAPs) and MT-associated proteins (MTAPs) because the isolation of actin as MFs and tubulin as MTs is a prerequisite for identifying MF-binding proteins (MFBPs), MTAPs, and any other cellular components that might associate in vivo with the CSK.

Light-induced synthesis of anthocyanin in carrot cells in suspension. IV: The action spectrum

Abstract— Using carrot cell suspension in 2,4‐dichlorophenoxyacetic acid (2,4‐D)‐depleted culture medium, fluence‐response curves for the formation of anthocyanin were determined at various wavelengths from 250 to 800 nm. In the fluence‐response curves at wavelengths between 260 and 330 nm, the response showed a sharp fluence‐dependent increase after the fluence exceeded threshold level at the respective wavelength. Such a sharp increase in response was not observed by light at 450 nm or longer wavelengths, although the response obtained by higher fluence of such light was always higher than that in the dark control. Action spectra determined at the sharp increasing phase of the response showed the single peak at 280 nm which equals the absorption maximum of UV‐B photoreceptor.

The plant cytoskeleton

Publisher Summary This chapter discusses the plant cytoskeleton. Even though the major constituents and the general filamentous and tubular structures of the plant cytoskeleton are similar to those of animals, there are numerous differences in their functions; many of these arise either from differences in the structure of individual cells in plants or from the architectural coherence of cells within multicellular plants. The basic structure of an animal cell entails a plasma membrane surrounding a dense cytoplasm containing the nucleus and all the other organelles, including the cytoskeleton, and the cytoplast is often coated with a thin extra-cellular matrix. In contrast, fully-enlarged plant cells consist of a plasma membrane surrounding a thin smear of cytoplasm, which, in turn, encloses a massive vacuole, while the plasma membrane is almost always enclosed in a tough cell wall.

Phenolic compounds in embryos of triticale caryopses at different stages of development and maturation in normal environment and after dehydration treatment

High performance liquid chromatography analysis revealed the presence of five phenolic acids in embryos of triticale caryopses (vanillic, caffeic, p-coumaric, ferulic and sinapic). Free phenolic acids reached the highest level at the early stages of development when germination was the lowest and decreased considerably in embryos at the final stage of grain maturation when germination was the highest. Revealing inverse correlation between the contents of free phenolic acids in developing embryos and intensity of precocious germination may indicate a role of phenolic acids in preventing pre-harvest sprouting in cereals. Total content of phenolic acids in embryos (i.e. free, and liberated from soluble esters and glycosides) increased gradually to 43DAF and decreased at full ripeness, whereas the content of total phenolics fluctuated slightly in embryos during development and ripening of caryopses. Enforced dehydration of unripe triticale caryopses stimulated germination both in embryos and whole grains. During the enforced dehydration treatment, a decrease in total content of both phenolic acids and phenolic compounds in embryos of triticale caryopses at different stages of development and maturation was observed. It should be stressed, that a number of naturally occurring phenolics are known to inhibit the germination of cereal grains. A possible role for phenolics in preventing pre-harvest sprouting and acclimation to dehydration in cereals is discussed.

Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)

Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3′ untranslated regions (3′-UTR). There are some similarities between the 3′-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3′-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.

Sub-cellular distribution and isotypes of a 49-kDa apyrase from Pisum sativum

Abstract We isolated a 49-kDa protein from various sub-cellular fractions from pea (Pisum sativum L. var. Alaska) stems using heparin affinity and cation exchange column chromatography. The corresponding proteins from all these fractions were identified as apyrase (EC 3.6.1.5) because they hydrolyzed both nucleoside tri- and diphosphates into their respective monophosphates. Using an antibody raised against apyrase, we studied the enzyme’s sub-cellular distribution in isolated fractions and found significant amounts in the cell wall (50%), the supernatant (33%), the cytoskeleton (14%), and the nuclei (3%). Immuno-electron microscopy using gold-labeled antibody confirmed that apyrase was present in cell walls, nuclei, and in filamentous structures in the cytoplasm associated with ribosomes. Even though there is only one gene (with two alleles), for this protein, 2D gels indicated there were at least five isotypes, three being major, and the relative abundance of these isotypes differed in different fractions. Enzymes from all fractions: (a) hydrolyzed nucleoside triphosphates and diphosphates, but not monophosphates, (b) were insensitive to most ATPase inhibitors (azide, fluoride, nitrate, molybdate, ouabain, quercetin), but (c) were all inhibited by vanadium pentoxide at relatively high concentrations. There were, however, some subtle differences between enzymes from different sub-cellular fractions, including different ADP/ATP hydrolysis ratios. These results show that the 49-kDa apyrase is located in various compartments within the cell (cell wall, nuclei, and the cytoskeleton) and that the enzymes from all fractions are basically similar in their apyrase function. We suggest that the enzyme is modified in various ways to furnish different forms with different (non-apyrase) functions in different sub-cellular locations.

Alternations in phenolic acids content in developing rye grains in normal environment and during enforced dehydration

A series of high pressure liquid chroamtography analyses revealed the presence of five phenolic acids in rye caryopses (vanillic, caffeic, p-coumaric, ferulic and sinapic), three of which (p-coumaric, ferulic and sinapic) were found in the free phenolic fraction. Ferulic acid was predominant, both among free acids and total phenolic acids (i.e. free, liberated from soluble esters and glycosides). The highest content of the free phenolic acids in rye caryopses was observed at the beginning of development, when on 22 DAF it was estimated at 11.55 µg·g−1 DW. During dehydratation the total level of free phenolic acids in rye caryopses decreased in all investigated samples. Although total phenolic acids contents in all samples of unripe rye caryopses always decreased after dehydration, in rye sample collected in full ripeness (57 DAF), the amount of these compounds increased after the enforced dehydration. It should be added that in ester-bound-soluble phenolic acids fraction (the largest part in the total phenolic acids fraction), irrespective of the total amount decrease, much increase of sinapic acid content in this fraction was observed after dehydratation treatment in all investigated samples of caryopses of various ripeness. During the development and ripening of rye caryopses, a gradual increase in the precocious germination ability of the grain was observed. The enforced dehydration stimulated the process of precocious germination of developing and ripening rye caryopses. A possible role of phenolics in preventing precocious germination and acclimation to dehydration of developing and ripening rye grains is discussed.

The influence of abscisic acid on different polysomal populations in embryonal tissue during pea seeds germination

The influence of abscisic acid (ABA) on the processes of formation of different polysomal populations, their structures and stability in embryonal tissue during pea seeds germination was studied. The contents of total ribosomal fraction increased in all samples up to 72 h of germination and then decreased. The contents of polysomal population (FP, MBP, CBP and CMBP) extracted from the embryonal tissue after 72 hrs of germination of pea seeds were then quantified. It turned out that in examined tissue of control sample, fraction of free polysomes (FP) was the most abounded. This population of polysomes in sprouts decreased after ABA treatment. FP content decreased even more when the higher ABA concentration was applied during germination. Similar changes were observed in the fraction of membrane-bound polysomes (MBP). Quite different tendencies were found, however, in forming population of the cytoskeleton-membrane-bound polysomes (CMBP). The CMBP population content in embryonal tissue increased in a dosage dependent manner with increasing concentration of ABA applied during seed germination. This indicates the important role of CMBP fraction in synthesis of specific proteins in embryos in the time when processes of seeds germination are retarded by ABA. In the final part we examined the stability of polysomes isolated from sprouts of germinating seeds in water and sprouts isolated from seeds treated with ABA (100 µM) during germination. Total polysomes isolated from embryonal tissue of germinating seeds treated with ABA showed much higher resistance to exogenous ribonuclease digestion than total polysomes of control sample. The obtained results suggest that ABA influence on different polysomal population formation also controls their stability.