Eric Davies

Intercellular communication in plants: electrical stimulation of proteinase inhibitor gene expression in tomato

Abstract. Tomato (Lycopersicon esculentum L.) plants accumulate proteinase inhibitor (u200apin) mRNA in response to various stimuli in leaves distant from those treated. Most earlier work suggests that the intercellular wound signals are chemical; we have tried to determine whether electrical or hydraulic signals can also evoke systemic pin expression. We used a mild flame to evoke a hydraulic signal and its local electrical aftermath, the variation potential (VP), and an electric stimulus to trigger an action potential. Under medium light, wounding evoked a 3- to 5-fold systemic increase in pin mRNA within 15 min, suggesting involvement of a rapidly transmitted signal. Wounding also triggered a transient systemic increase in calmodulin (calu200a) mRNA, under medium light conditions. Wounded plants exhibited electrical responses (VP) and yielded 5-to 15-fold increases in pin mRNA within 1 h. Electrically stimulated plants that transmitted an action potential to the analyzed leaf exhibited similarly large, rapid increases in pin transcript. Plants which generated no signal had unchanged levels of pin mRNA. Thus, in addition to the previously shown chemical signals, both hydraulically induced VPs and electrically induced action potentials can elicit systemic pin expression.

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.

Isolation of F-actin from pea stems

SummaryA procedure is introduced which allows the isolation of abundant amounts of F-actin from plants (etiolated pea seedlings) in an array of morphologies very similar to the array of morphologies found in situ. The major feature is a homogenizing medium containing very low ionic strength, low monovalent ion (K+) concentration, a 3-fold higher level of Mg+ +, the presence of EGTA to chelate Ca++, and PMSF to inhibit protease activity. Using this buffer, about 80–90% of the sedimentable actin is found in the low speed (4,000×g) pellet.

Protein bodies in corn endosperm are enclosed by and enmeshed in F-actin

SummaryWhen corn endosperm is gently homogenized in a microfilament-stabilizing buffer and filtrates centrifuged at very low g forces, abundant protein bodies are present in the pellet. When stained with both rhodamine-phalloidin and fluorescein-S1-myosin and viewed under epifluorescence, individual protein bodies appear to be enclosed in F-actin and groups of protein bodies appear to be enmeshed within a network of fine actin filaments. The yield of actinenclosed protein bodies is not affected by the addition of non-ionic detergents to the grinding medium, but it is severely decreased with the addition of cytoskeleton-disrupting agents including Tris-HCl, KCl, KI, and ammonium sulphate, and the interconnecting fine filaments are totally abolished. Heparin, which interacts with membrane-cytoskeleton complexes, causes apparent coagulation of the protein bodies. The evidence from fluorescent microscopy suggesting that F-actin surrounds and enmeshes the protein bodies is supported by electron microscopy, gel electrophoresis, and Western blot analysis.

Cytoskeleton‐bound polysomes in plants. III. Polysome‐cytoskeleton‐membrane interactions in corn endosperm.

Over 80% of the polysomes in corn endosperm sediment along with protein bodies at 30 xg from seeds ground in cytoskeleton‐stabilizing buffer. The cytoskeleton‐disrupting agents, Tris‐HCl, K+, heparin, and sodium deoxycholate cause polysome release, while protease K and the non‐ionic detergent, PTE, are effective only in the presence of these agents, and RNase is almost without effect. We suggest that many of the polysomes in corn endosperm are associated via their ribosomes, but not mRNA or nascent polypeptides with the actin component of the cytoskeleton and only indirectly with membranes. Corn endosperm homogenates examined under the fluorescence microscope show polysomes coating individual protein bodies and co‐localizing with actin, but not with ER.

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.

Co-localization of polysomes, cytoskeleton, and membranes with protein bodies from corn endosperm

SummaryFrozen corn endosperm was homogenized in a cytoskeleton-stabilizing buffer and stained directly (without pelleting) with rhodamine-phalloidin for actin and either thiazole orange to stain RNA or DiOC6 to stain membranes prior to examination under the fluorescence microscope. Other samples were treated with a non-ionic detergent alone or in conjunction with a ionic detergent prior to staining and fluorescence microscopy. Very gentle homogenization in unsupplemented buffer yielded a massive aggregate containing protein bodies that fluoresced after treatment with the ER stain DiOC6. This aggregate was capped by an aggregate of unstained starch grains. More vigorous homogenization yielded more disperse patterns showing almost identical co-localization of ER, actin and RNA (polysomes). Homogenization in buffer plus non-ionic detergent removed most of the membrane yet maintained co-localization of actin and polysomes, while homogenization in double detergent removed the last traces of membrane and actin, and released over 70% of the polysomes. We interpret these results to suggest that protein bodies are surrounded by membranes, cytoskeleton and RNA (polysomes) and that the majority of the polysomes are attached more firmly to the cytoskeleton than to the membrane. This provides evidence from fluorescence microscopy to supplement that from biochemical analyses for the existence of cytomatrix-bound polysomes in plants.

Polyribosome degradation as a sensitive assay for endolytic messenger-ribonuclease activity.

Abstract A method is described whereby picogram amounts of pancreatic RNase can be accurately measured using polyribosomes as substrate. The method involves incubating isolated polysomes in RNase, separating them on gradients, and calculating the number of interribosomal bonds for each profile. The decrease in number of interribosomal bonds is proportional to the amount of enzyme added from 20 to 200 pg of pancreatic RNase per assay when conducted at 2°C for 20 min. Sensitivity can be increased by increasing the temperature or the duration of the incubation. Since the method of analysis of the reaction product is unaffected by exonuclease activity, the assay is specific for endonucleases. It can, therefore be used to assay for the presence of endonucleases as contaminants in preparations of exonucleases and to determine the mode of action of nucleases. The method has been used to measure the amount of soluble and polysome-associated RNase in pea stem tissue.

A potential role for cellulase in hormone-controlled elongation

Summary Measurements were made of the effects of indoleacetic acid (IAA), gibberellic acid (GA) and cycloheximide (CH) on growth, protein content and cellulase activities of segments derived from the apex of decapitated, etiolated epicotyls of Pisum sativum L. var. Alaska. Increasing amounts of CH caused increasing inhibition of both endogenous and hormonestimulated elongation, whereas certain critical levels of CH converted the expansion elicited by high levels of IAA into elongation. This CH-evoked conversion from expansion into elongation was accompanied by a partial decrease in protein accumulation and cellulase activity. Cellulase activity per segment increased in proportion to total growth (fresh weight) in tissues elongating in response to high levels of GA or low levels of IAA. In contrast, cellulase activity per segment rose markedly in tissues expanding in response to high levels of IAA. It is concluded that the amount of IAA present in the tissue governs the level of certain growth-limiting-proteins (GLP). These GLP, in turn, control the amount and direction of growth (i.e., elongation or expansion). It is suggested that cellulase should be considered as a candidate for the role of a GLP whose activity is modulated by IAA.

EFFECT OF LIGHT AND TRITON X-100 ON THE SIZE OF PROTOPLASTS FROM Ulothrix gigas

Abstract— An electronic particle‐size analyzer (Coulter Counter with Channelyzer) was used both to monitor the purity of large numbers of protoplasts during sequential steps in their isolation from Ulothrix gigas and also to detect changes in volume (swelling or growth) after incubation of purified protoplasts under various conditions.