Sieve Element Protoplast Research Articles

Identification of sieve elements and companion cell protoplasts by a combination of brightfield and fluorescence microscopy.

Premise of the StudyPhloem‐limited diseases are becoming increasingly pervasive, threatening the existence of crops worldwide. Studies of phloem diseases are complicated by the inaccessibility of the phloem tissue. Phloem cells are located deep inside the plant body, are interspersed with other cell types, are among the smallest cells in the plant kingdom, and make up a small percentage of the total cell population in a plant. These properties make phloem cells difficult to investigate.MethodsWe used leaf midrib protoplasts and a combination of organelle‐specific dyes including Neutral Red (acidic compartments), MitoTracker Green (mitochondria), Hoechst 3342 (nucleus), and chloroplast autofluorescence. We examined the protoplasts under light and fluorescence microscopy.ResultsWhen observed using brightfield and fluorescence microscopy, sieve element protoplasts were distinguished by the lack of vacuole and a nucleus, and by containing various mitochondria. Companion cells showed a dense cytoplasm and various small vacuoles. They also revealed their characteristic large nucleus and abundant mitochondria.DiscussionWe present evidence that a combination of organelle‐specific dyes and autofluorescence allows for the identification of sieve elements and companion cell protoplasts from citrus leaf tissue.

Electrophysiological approach to determine kinetic parameters of sucrose uptake by single sieve elements or phloem parenchyma cells in intact Vicia faba plants.

Apart from cut aphid stylets in combination with electrophysiology, no attempts have been made thus far to measure in vivo sucrose-uptake properties of sieve elements. We investigated the kinetics of sucrose uptake by single sieve elements and phloem parenchyma cells in Vicia faba plants. To this end, microelectrodes were inserted into free-lying phloem cells in the main vein of the youngest fully-expanded leaf, half-way along the stem, in the transition zone between the autotrophic and heterotrophic part of the stem, and in the root axis. A top-to-bottom membrane potential gradient of sieve elements was observed along the stem (−130 mV to −110 mV), while the membrane potential of the phloem parenchyma cells was stable (approx. −100 mV). In roots, the membrane potential of sieve elements dropped abruptly to −55 mV. Bathing solutions having various sucrose concentrations were administered and sucrose/H+-induced depolarizations were recorded. Data analysis by non-linear least-square data fittings as well as by linear Eadie–Hofstee (EH) -transformations pointed at biphasic Michaelis–Menten kinetics (2 MM, EH: Km1 1.2–1.8 mM, Km2 6.6–9.0 mM) of sucrose uptake by sieve elements. However, Akaike's Information Criterion (AIC) favored single MM kinetics. Using single MM as the best-fitting model, Km values for sucrose uptake by sieve elements decreased along the plant axis from 1 to 7 mM. For phloem parenchyma cells, higher Km values (EH: Km1 10 mM, Km2 70 mM) as compared to sieve elements were found. In preliminary patch-clamp experiments with sieve-element protoplasts, small sucrose-coupled proton currents (−0.1 to −0.3 pA/pF) were detected in the whole-cell mode. In conclusion (a) Km values for sucrose uptake measured by electrophysiology are similar to those obtained with heterologous systems, (b) electrophysiology provides a useful tool for in situ determination of Km values, (c) As yet, it remains unclear if one or two uptake systems are involved in sucrose uptake by sieve elements, (d) Affinity for sucrose uptake by sieve elements exceeds by far that by phloem parenchyma cells, (e) Patch-clamp studies provide a feasible basis for quantification of sucrose uptake by single cells. The consequences of the findings for whole-plant carbohydrate partitioning are discussed.

Solute concentrations of the phloem and parenchyma cells present in squash callus

Abstract. Glutaraldehyde fixation was used to determine the solute concentrations in the various cell types present in tissue cultures of squash (Cucurbita pepo). Small pieces of callus were plasmolyzed in a graded series of mannitol solutions and fixed in 20 kg m−3 glutaraldehyde adjusted to be isosmotic with the particular plasmolysing solution. The callus samples were further processed using standard electron microscopy techniques. Using this procedure, mature sieve elements that form in squash callus have an osmotic potentional of ‐2.4MPa. The osmotic potential of the callus sieve elements was comparable to values reported for the sieve tube members of the phloem in intact plants. This ability of callus sieve elements to develop high internal hydrostatic pressures demonstrates that they are capable of phloem loading. However, the osmotic potentials of the surrounding parenchymatous cells and companion cells were only –1.15 and –1.5 MPa, respectively. In contrast to the companion cells of the phloem in intact plant tissues, the osmotic potential of the callus companion cells indicated that they were not directly involved in phloem loading. Several immature sieve elements containing distinct nuclei and vacuoles were observed in the callus granules. These immature sieve elements were plasmolyzed in weaker mannitol solutions (below 0.6kmol m−3) than the enucleate sieve elements (1.01 kmol m−3 mannitol). The low solute concentrations in immature sieve elements indicated that the ability to load sugars occurs concomitantly with the maturation of the sieve element protoplast.

Ultrastructural studies of protophloem sieve elements in Gossypium hirsutum

The differentiation of sieve element protoplasts was studied in the root protophloem of Gossypium hirsutum (cotton). The nuclei degenerate, but frequently remain into late stages of sieve element maturation as darkly stained amorphous aggregates. At the beginning of nuclear degeneration the chromatin aggregates, usually near the envelope, and becomes a composite of darker and lighter stained granular material. Membranous material of unknown origin is also frequently associated with the chromatin. The protophloem sieve element nucleolus is smaller than nucleoli in adjacent parenchyma cells and persists into the intermediate stages of nuclear degeneration. When the nuclear envelope is still intact, the endoplasmic reticulum (ER) cisternae form a small number of stacks, some of which may lie near the nucleus. The peripheral regions of protophloem sieve elements are conspicuously deficient in aggregated ER. Convoluted membranous material, apparently of ER origin, accumulates near the nuclear envelope. Protophloem sieve element plastids lack the characteristic starch grains in early stages of differentiation, but usually develop one or more grains at a somewhat later stage in differentiation. Mitochondria decrease in density as the cell matures and frequently become associated with ER. Plastids and mitochondria become incorporated in the stacked and convoluted ER that accumulates near the nuclear envelope. In mature sieve elements the plastid starch grains become fissured and coarsely granular.

Characteristics of Structure and Differentiation in the Sieve Element of Lower Vascular Plants

AbstractThe structure and differentiation of the sieve element of lower vascular plants is reviewed using data obtained primarily from ultrastructural investigations conducted during the last ten years. During the last decade the phloem of representatives from every major group of the ferns and fern allies has been examined with the electron microscope and from these studies a rather clear picture has emerged of the structure of the sieve element protoplast in this diverse group of plants. Present data indicate that although the details of sieve‐element differentiation may differ, the protoplasts of the mature sieve elements in the various groups of lower vascular plants are remarkably similar in structure. Each consists of a plasmalemma, a parietal, anastomosing network of smooth ER, plastids, mitochondria and, with the exception of the lycopods, variable numbers of refractive spherules. The protoplasts of mature sieve elements are joined by plasmalemma‐lined connections, each arising from a single plasmodesma during the course of sieve element differentiation. The size of the connections in the mature elements range from plasmodesmata‐like structures to relatively wide sieve‐area pores, depending on the species. Moreover, the contents of the cytoplasmic connections vary somewhat according to the species. Whereas in the lycopods, the sieve‐area pores are virtually unoccluded by any cytoplasmic material, the cytoplasmic connections of all other lower vascular plants examined with the electron microscope contain variable amounts of membranous material, apparently tubular elements of ER. In Equi‐setum hyemale, Psilotum nudum and the eusporangiate and protoleptosporangiate ferns, the ER membranes are very numerous and virtually occlude the pores. Furthermore, the membranes apparently are not connected with the parietal ER in the lumen of the cell. The sieve‐area pores of the leptosporangiate ferns also contain ER membranes, however, they are not as abundant as the membranes of the eusporangiate and protoleptosporangiate ferns. In addition, in the leptosporangiate ferns the pore membranes apparently are united with the parietal ER in the lumen of the cell.

Nucleus and endoplasmic reticulum in differentiating root protophloem of Nicotiana tabacum

In protophloem sieve elements of roots of Nicotiana tabacum L., nuclei disintegrate completely during cell differentiation. While the sieve element still hardly differs from meristematic cells next to it, the nucleus shows indications of reduction in the amount of chromatin, and its nucleolus is small. After the contents of the nucleus are more or less completely eliminated, the nuclear envelope partly collapses. Its integrity breaks down and the nuclear space is invaded by cytoplasmic ribosomes. The endoplasmic reticulum (ER) increases in amount as differentiation progresses and the initially scattered cisternae are assembled into stacks. Electron-dense material appears between the cisternae of a stack. Development of stacked ER is at maximum just before reorganization of the protoplast is completed and the cell reaches the mature state. A small amount of ER is present in the mature cell, but the nucleus is absent. Various other phenomena of specialization of the sieve element protoplast are correlated with the changes in the nucleus and ER. Particularly notable is the timing in maturation of the sieve plate. While the nucleus collapses, the pore sites are still covered with ER cisternae. Removal of the ER from pore sites and the final opening of pores occur at the end of differentiation of the sieve element.

Movement of Sugars through Plants by Cytoplasmic Pumping

MUNCH'S hypothesis1 for the mechanism of translocation in plants depends on a pressure gradient in sieve tubes driven by osmosis. It is supported by mass flow movements of mobile compounds2,3 in the phloem. Other hypotheses4–6 suggest that the transport mechanism requires a direct supply of energy from metabolism; they are supported by the demonstration of high levels of respiration in the phloem7, and the sensitivity of phloem transport to light8, temperature9 and metabolic inhibitors10. This apparently conflicting evidence is reconciled in hypotheses by Fensom11 and Spanner12, who independently proposed electrokinetic mechanisms which involve metabolic pumping at sieve plates and a mass flow of solution in the lumina of sieve elements. Each hypothesis implies a particular structure of the sieve element protoplast, but cytological studies are at present inconclusive because some indicate a degradation of the protoplast of sieve elements while others suggest a specialized organization of cytoplasm. In this article I wish to propose that sugars and other solutes are pumped over long distances through plants by transcellular strands of cytoplasm in sieve tubes.

Tubular and fibrillar components of mature and differentiating sieve elements.

An ontogenetic study of the sieve element protoplast of Nicotiana tabacum L. by light and electron microscopy has shown that the P-protein component (slime) arises as small groups of tubules in the cytoplasm. These subsequently enlarge to form comparatively large compact masses of 231 +/- 2.5 (SE)A (n = 121) tubules, the P-protein bodies. During subsequent differentiation of the sieve element, the P-protein body disaggregates and the tubules become dispersed throughout the cell. This disaggregation occurs at about the same stage of differentiation of the sieve elements as the breakdown of the tonoplast and nucleus. Later, the tubules of P-protein are reorganized into smaller striated 149 +/- 4.5 (SE)A (n = 43) fibrils which are characteristic of the mature sieve elements. The tubular P-protein component has been designated P1-protein and the striated fibrillar component P2-protein. In fixed material, the sieve-plate pores of mature sieve elements are filled with proteinaceous material which frays out into the cytoplasm as striated fibrils of P2-protein. Our observations are compatible with the view that the contents of contiguous mature sieve elements, including the P-protein, are continuous through the sieve-plate pores and that fixing solutions denature the proteins in the pores. They are converted into the electron-opaque material filling the pores.

Tubular components in cells of healthy and tobacco mosaic virus-infected Nicotiana

The protein component (slime) of the sieve element protoplast in Nicotiana tabacum L., here designated as P-protein (phloem protein), consists of tubular elements, at least while the protein occurs in the form of discrete bodies. In this form the P-protein has a striking resemblance to one of the tubular components, here designated as X-component, occurring in tobacco cells infected with tobacco mosaic virus (TMV). The X-component may be differentiated from the P-protein by the straight form of the tubules, the greater degree of order in the arrangement of the tubules in the aggregates, and to some extent by size. The X-component occurs also in other forms, but all these forms may be arranged in a series suggesting a developmental sequence. The sequence seems to be from filaments composed of groups of tubules to individual tubules assembled in larger groups with some order in the arrangement. It is suggested that the X-component may be the noninfectious protein, or X-protein, commonly associated with complete virus particles in TMV-infected tobacco.

Cytoplasm Exudate from Cut Phloem

CYTOPLASM structure in sieve elements and particularly at sieve pores is of critical importance in any consideration of the translocation mechanism. The mass flow theory1 assumes a degenerate sieve element protoplast in which vacuolar fluid fills the sieve element and is continuous through open sieve pores. A fluid flow of sucrose solution is thought to occur through sieve tubes due to an osmotic pressure gradient. Other theories propose that metabolic energy provides the motive force required to move nutrients over long distances through plants. These ‘active’ theories assume that the sieve elements have cytoplasmic contents similar to parenchyma cells but with exceptionally large end wall pores which are filled with cytoplasm. This interpretation is consistent with de Vries protoplasmic streaming theory2, with Mason and Maskell's activated diffusion theory3, with Kursanov's molecular transfer theory4 and Spanner's electrokinetic theory5.

Callose Substance in Sieve Elements

The secondary phloem of 6 species of woody dicotyledons was examined for the occurrence of callose on the sieve plates of active sieve elements. Fluorescence and bright-field staining methods were used to detect callose. Tissue from the 6 species was killed and fixed in each of 5 solutions. Some tissue of each species was submerged in the killing solutions as quickly as possible, the remainder within 15 min after removal from the tree. In each species, some active sieve elements of the quick-killed tissue gave negative callose reactions. All active sieve elements of the delay-killed tissue gave positive callose reactions. These and other results suggest that the active sieve elements in the secondary phloem of the species studied normally lack callose and that the extent of callose deposition in these cells depended primarily upon the rapidity with which the sieve-element protoplasts were killed after wounding of the phloem. In addition, bright-field observations of sieve plates of large numbers of sieve elements from a seasonal collection of Tilia americana secondary phloem suggest that the active sieve elements normally lack callose during the growing season and that the inactive sieve elements normally possess it (dormancy callose).