Eldon H. Newcomb

Fine-structural characterization of plant microbodies.

SummaryMorphology and distribution of the relatively less well known organelles of plants have been studied with the electron microscope in tissues fixed in glutaraldehyde and postfixed in osmium tetroxide. An organelle comparable morphologically to the animal microbody and similar to the plant microbody isolated by Mollenhauer et al. (1966) has been encountered in a variety of plant species and tissues, and has been studied particularly in bean and radish roots, oat coleoptiles, and tobacco roots, stems and callus. The organelle has variable shape and is 0.5 to 1.5 μ in the greatest diameter. It has a single bounding membrane, a granular to fibrillar matrix of variable electron density, and an intimate association with one or two cisternae of rough endoplasmic reticulum (ER). Microbodies are easily the most common and generally distributed of the less well characterized organelles of plant cells. It seems very probable that they contain the enzymes characteristic of animal lysosomes (containing hydrolases) or animal microbodies (containing catalase and certain oxidases). Spherosomes are also possible sites of enzyme activity but are not as common or as widely distributed as microbodies. For this reason it appears likely that the particles designated as “plant lysosomes”, “spherosomes”, “peroxisomes”, etc., in some of the cytochemical and biochemical studies on enzyme localization will prove to be microbodies.Variations in the morphology and ER associations of microbodies in tissues of bean and radish are described and discussed. “Crystal-containing bodies” (CCBs) are interpreted as a specialized type of microbody characteristic of metabolically less active cells. Stages in the formation of CCBs from microbodies of typical appearance are illustrated for Avena.The general occurrence of microbodies in meristematic and differentiating cells and their close association with the ER suggest that they may play active roles in cellular metabolism. The alterations in their morphology and numbers that are observed in certain differentiating cells suggest further that the enzyme complements and metabolic roles of microbodies might change during cellular differentiation. If so, microbodies could be the functional equivalent of both microbodies and lysosomes of animal cells.

Coated vesicles and other cytoplasmic components of growing root hairs of radish

SummaryRoot tips of radish,Raphanus sativus, were fixed in glutaraldehyde followed by osmium tetroxide. The fine structure of young root hairs, not exceeding about 130μ, in length, was studied to relate their apical growth pattern to their cytoplasmic organization. The cytoplasm in the terminal 3–5μ it of the root hair is characterized by an electron dense matrix in which lie numerous smooth-surfaced vesicles, large irregularly-shaped fibrous inclusions, and clusters of ribosomes. Other organelles are largely or entirely excluded from this region. Farther than about 5μ, from the tip, the hair cytoplasm is filled with plastids, rough endoplasmic reticulum, mitochondria, and dictyosomes. The latter produce smooth vesicles similar in size and morphology to those present in the apical dome. Vesicles of a different kind appear in the peripheral cytoplasm along the entire length of the hair. These vesicles possess an alveolate or chambered coat about 20 mμ thick and have a diameter of about 85 mμ, including coat. They originate by evagination from the large, smooth-surfaced vesicles in the vicinity of dictyosomes. It is suggested that proteins and carbohydrates are concentrated in the dictyosomes and then segregated in the smooth vesicles released from the dictyosome cisternae. The coated vesicles which bud from the smooth vesicles may serve to isolate the proteins and transport them to the hair surface for participation in wall synthesis. The smooth vesicles are believed to convey carbohydrates to the region of active wall extension at the hair apex.

Microbody-like organelles in leaf cells.

An organelle approximately 0.5 to 1.5 microns in diameter, limited by a single membrane, occurs abundantly in the chlorophyllous cells of leaves of several dicotyledonous and monocotyledonous plants. Its finely granular matrix frequently contains crystalline, fibrous, or amorphous inclusions. It is frequently appressed to a chloroplast or squeezed between chloroplasts so that its limiting membrane is in extensive contact with the outer membranes of the chloroplast envelopes. The organelle is probably identical with recently isolated leaf particles that contain enzymes involved in the metabolism of glycolate, a chloroplast product; it is interpreted as a form of plant microbody.

Fine structural investigation of nuclear inclusions in plants.

Light microscopic observations dating back to 1859 have established that nuclear inclusions occur widely in plant and animal cells. The present fine structural investigation describes nuclear inclusions in thirteen species of plants representing ferns, a gymnosperm, and angiosperms. Ultrastructurally the inclusions are quite diverse and include several morphologically distinct types represented by crystalline, fibrous, or amorphous structures. In some species the inclusions commonly attain lengths of several microns and occupy as much as 20–40 % of the median nuclear cross sectional area. In several species inclusions identical to those in the nucleus have been encountered in the perinuclear space and cytoplasm. Histochemical staining indicates that most of the inclusions are proteinaceous, although spherosome-like bodies presumably consisting of lipid have been observed also. Dissimilarities between the proteinaceous inclusions described here and inclusions observed previously in virus-infected plants are summarized. In many species the inclusions appear to be related to the nucleoli, either through close association or through an inverse size relationship shown during development. In a fern, observations made during cell differentiation indicate that the appearance of nuclear inclusions may coincide with dissolution of the nucleolus. Attention is directed to the possible similarity between the formation of nuclear inclusions in plants and the segregation of material from the nucleolus in animal tissues treated with actinomycin D.

Fine structure of cell plate formation in the apical meristem of Phaseolus roots

The fine structure of dividing root tip cells of Phaseolus vulgaris L. has been studied in material fixed in glutaraldehyde followed by osmium tetroxide and poststained in uranyl acetate and lead citrate. Microtubules, observed in both longitudinal and transverse sections of the root tip, are abundant during the early stages of cell plate formation when vesicles from the dictyosomes are aggregating and beginning to fuse in the plate region, but disappear in later stages during which fusion continues and the vesicular aggregates grow laterally. The distribution of microtubules among the vesicles is observable in transverse sections in which the young plate is seen in face view. No consistent grouping of microtubules around the vesicles or structural association between microtubules and vesicles has been detected. Analysis of progressive stages of plate development made from transverse sections reveals that the fusing vesicles subsequently form conspicuous stellate or branched bodies, the prominent linear arms of which protrude at sharp angles from the central mass. It appears that the arms are the sites of further fusion and growth, and that the growth is confined to the plane of the plate. In later stages the branched bodies continue to grow and fuse, creating large interconnected expanses of cell plate material.

A correlative ultrastructural and enzymatic study of cotyledonary microbodies following germination of fat-storing seeds.

SummarySunflower, cucumber, and tomato cotyledons, which contain microbodies in both the early lipid-degrading and the later photosynthetic stages of post-germinative growth, were processed for electron microscopy according to conventional procedures and examined 1, 4 and 7 days after germination. Homogenates of sunflower cotyledons were assayed for enzymes characteristic of glyoxysomes and leaf peroxisomes (both of which are defined morphologically as microbodies) at stages corresponding to the fixations for electron microscopy. The particulate nature of these enzymes was demonstrated by differential and equilibrium density centrifugation, making it possible to relate them to the microbodies seen in situ.One day after germination, the microbodies are present as small organelles among large numbers of protein and lipid storage bodies; the cell homogenate contains catalase but no detectable isocitrate lyase (characteristic of glyoxysomes) or glycolic acid oxidase (characteristic of leaf peroxisomes). 4 days after germination, numerous microbodies (glyoxysomes) are in extensive and frequent contact with lipid bodies. The microbodies often have cytoplasmic invaginations. At this stage the cells are rapidly converting lipids to carbohydrates, and the homogenate has high isocitrate lyase activity. 7 days after germination, microbodies (peroxisomes) are appressed to chloroplasts and frequently squeezed between them in the green photosynthetic cells. The homogenate at this stage has substantial glycolic acid oxidase activity but a reduced level of isocitrate lyase. It is yet to be determined whether the peroxisomes present at day 7 are derived from preexisting glyoxysomes or arise as a separate population of organelles.

Ultrastructure and distribution of microbodies in leaves of grasses with and without CO2-photorespiration.

SummaryA comparative study was made of the ultrastructure, distribution and abundance of leaf microbodies in four species of “temperate” grasses with high and four “tropical” grasses with low CO2-photorespiration. The temperate grasses were all festucoid; the tropical grasses included two panicoid species and two chloridoid. Comparisons of relative abundance were made by computing the average numbers of microbody profiles per cell section.Although microbodies were present in the green parenchymatous leaf cells in all grasses examined, their average number per cell was in general severalfold greater in the grasses with high CO2-photorespiration than in those with low. Furthermore, whereas in the grasses with high CO2-photorespiration the microbodies were distributed through the mesophyll, in those with low CO2-photorespiration they were concentrated in the vascular-bundle-sheath cells and were smaller and relatively scarce in the mesophyll cells. The leaf microbodies of the eight grass species resembled one another in general morphology, but differed to some extent in regard to size and type of inclusion. Microbodies of all four festucoid species contained numerous fibrils with a discernible substructure. Those of the two panicoid species contained clusters of round bodies with transparent cores. The equivalence of the microbodies to peroxisomes as biochemically defined was shown cytochemically by employing 3,3′-diaminobenzidine for the localization of catalase, a marker enzyme for the peroxisome. This reaction was blocked by the catalase inhibitor, aminotriazole.The observations on the relative abundance and distribution of peroxisomes in leaves of grasses with high CO2-photorespiration versus those with low are consistent with the published biochemical data on the levels and distribution of peroxisomal enzymes in representatives of plants with high and low CO2-photorespiration, and may help explain the differences in apparent photorespiratory levels between these two groups of plants.

The ultrastructure and development of tubular and crystalline P-protein in the sieve elements of certain papilionaceous legumes

SummaryThe ultrastructure of the primary sieve elements of several papilionaceous legumes was studied using hypocotyl and young internode segments fixed in glutaraldehyde followed by osmium tetroxide. In particular, the study sought to determine whether the crystalline “flagellar inclusions” characteristic of these species are developmentally related to the P-protein bodies present in the phloem of these and other legumes and of angiosperms generally. The crystalline inclusions consist of a central body terminated at one or both ends by a gradually tapering tail. The central body is usually spindle shaped in longitudinal section and square in cross section. In all species examined, the inclusion is first seen as a small, thin crystal in the cytoplasm of young sieve elements. The crystal enlarges and acquires tails as the sieve element develops. In certain species, exemplified byDesmodium canadense, numerous tubules are formed in the cytoplasm near the crystal and appear to be concerned in its growth. The observations on the structure and interactions of these two components, tubules and crystalline inclusions, suggest that both represent forms of P-protein: the tubules are continuous with the crystal and are striated like the crystal near the tubule-crystal junction, suggesting that they are adding onto the crystal body; the tubules closely resemble the P-protein tubules described in the literature in that they measure 157 Å in diameter, accumulate in spindle-shaped bundles, and disperse into striated fibrils late in the ontogeny of the sieve element; and finally, the crystal also disperses into fine filaments. The crystalline inclusion therefore probably represents still another aggregation state of P-protein, one that is characteristic of papilionaceous legumes. The different stages of crystal aggregation and the diverse forms of P-protein now known are discussed briefly in relation to the control of macromolecular assembly and subunit packing.

Immunogold localization of nodule-specific uricase in developing soybean root nodules

Immunogold labeling was used to study the time of appearance and distribution of a nodule-specific form of uricase (EC 1.7.3.3) in developing nodules of soybean (Glycine max (L.) Merr.) inoculated with Bradyrhizobium japonicum. The enzyme was detected in thin sections of tissue embedded in either L R White acrylic resin or Spurrs epoxy resin, by employing a polyclonal antibody preparation active against a subunit of soybean nodule uricase. Antigenicity was better preserved in L R White resin, but ultrastructure was better maintained in Spurrs. Uricase was first detectable with protein A-gold in young, developing peroxisomes in uninfected cells, coincident with the release of Bradyrhizobium bacteroids from infection threads in adjacent infected cells. As the peroxisomes enlarged, labeling of the dense peroxisomal matrix increased. Gold particles were never observed over the paracrystalline inclusions of peroxisomes, however. Despite a close association between enlarging peroxisomes and tubular endoplasmic reticulum, uricase was not detectable in the latter. In mature nodules, labeling of uricase was limited to the large peroxisomes in uninfected cells. Small peroxisome-like bodies present in infected cells did not become labeled.

Formation and dispersal of crystalline P-protein in sieve elements of soybean (Glycine max L.)

SummaryLight microscopic observations dating back to 1892 have established that sieve elements of papilionaceous legumes contain a unique type of slime body. This large, compact crystalline type of P-protein has also been observed in sieve elements in recent electron microscopic investigations but its formation and possible relationship to other P-protein structures have not been examined. The present fine structural study describes its development in hypocotyl tissue of 4-day old seedlings of soybean (Glycine max L.). Preceding the formation of a P-protein body, a young sieve element possesses large numbers of ribosomes, abundant vesiculate ER and numerous dictyosomes surrounded by vesicles. A finely granular material accumulates among these components, then condenses into electron opaque masses. Scattered bundles of tubules appear within these masses, then aggregate, and next align longitudinally in the sieve element. By a further transformation, the tubules are converted into an electron opaque crystalline P-protein body. This body continues to grow by aggregation and transformation of additional tubules, and at maturity may be as long as 15–30 microns. The main body, which is square in cross section, tapers toward the ends and is terminated by sinuous “tails”. Eventually this crystal disperses into a mass of fine striated fibers that fills the lumen of the mature sieve element. Attention is directed to similarities between the bundles of tubules and previously described “extruded nucleoli”. Factors possibly involved in the structural variations and transformations described above are also discussed.