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).     

Sieve Element of Impatiens Sultanii: 2. Developmental Aspects

The sieve elements arise from vacuolate cells, and enlargementof one or more slime bodies increases the volume of cytoplasmrelative to that of the vacuole. The slime finally dispersesthroughout the region once occupied by the vacuole. A new term,mictoplasm, is proposed for the resulting mixture of non-membranouscytoplasmic material, including slime, with the contents ofthe vacuole. The nucleus disappears during development, butbefore losing its chromaticity, it apparently releases one ormore nucleoli into the cytoplasm. The extruded nucleoli areprominent during development but usually disappear with thetonoplast, nucleus, and dictyosomes as the cell matures. Atmaturity, small vesicles, plastids containing spherical starchgrains, and sparsely distributed mitochondria deficient in tubulesare attached to the plasmalemma. The sieve-plate connectingstrand develops in a pore site bearing a pair of callose plateletsand penetrated in the centre by a plasmodesms. The callose cylinderwhich surrounds the mature connecting strand is followed sothat the shape of a connecting strand in cross section is stellate.Mictoplasm and the plasmalemma are continuous from one cellto the next through the sieve-plate connecting strands.     

Unplugging the callose plug from sieve pores

The presence of callose in sieve plates has been known for a long time, but how this polysaccharide plug is synthesized has remained unsolved. Two independent laboratories have recently reported the identification of callose synthase 7 (CalS7), also known as glucan synthase-like 7 (GSL7), as the enzyme responsible for callose deposition in sieve plates. Mutant plants defective in this enzyme failed to synthesize callose in developing sieve plates during phloem formation and were unable to accumulate callose in sieve pores in response to stress treatments. The mutant plants developed less open pores per sieve plate and the pores were smaller in diameter. As a result, phloem conductivity was reduced significantly and the mutant plants were shorter and set fewer seeds.Key words: Arabidopsis thaliana, callose, callose synthase, glucan synthase-like, phloem, plasmodesmata, sieve plate     

Auxin promotes dormancy callose removal from the phloem ofMagnolia kobus and callose accumulation and earlywood vessel differentiation inQuercus robur

During winter, the phloem of the diffuse-porous tree magnolia (Magnolia kobus DC.) is dormant and is characterized by heavy deposits of dormancy callose. Application of 1-naphthaleneacetic acid (NAA) to either the top or the lower ends of excised dormant branches before bud break resulted in the removal of the dormancy callose from the sieve tubes. In both intact and auxintreated branches, callose degradation occurred first in the recently formed sieve tubes. There was no new vessel differentiation in magnolia before bud break. In contrast, the sieve tubes of the ring-porous oak (Quercus robur L.), which possess massive dormancy callose deposits during winter, were almost callose-free just before bud break. Application of auxin to the top of excised branches before bud break resulted in callose accumulation on the most recently formed sieve tubes. The first earlywood vessels were evident in oak before bud break, and their numbers were increased by auxin application. The early development of phloem and xylem (before bud break) in ring-porous species is an ecological adaptation which prepares the vascular system of these trees to function immediately at the beginning of their growing season which is relatively short.     

Silicon amendment to rice plants contributes to reduced feeding in a phloem‐sucking insect through modulation of callose deposition

Silicon (Si) uptake by Poaceae plants has beneficial effects on herbivore defense. Increased plant physical barrier and altered herbivorous feeding behaviors are documented to reduce herbivorous arthropod feeding and contribute to enhanced plant defense. Here, we show that Si amendment to rice (Oryza sativa) plants contributes to reduced feeding in a phloem feeder, the brown planthopper (Nilaparvata lugens, BPH), through modulation of callose deposition. We associated the temporal dynamics of BPH feeding with callose deposition on sieve plates and further with callose synthase and hydrolase gene expression in plants amended with Si. Biological assays revealed that BPH feeding was lower in Si‐amended than in nonamended plants in the early stages post‐BPH infestation. Histological observation showed that BPH infestation triggered fast and strong callose deposition in Si‐amended plants compared with nonamended plants. Analysis using qRT‐PCR revealed that expression of the callose synthase gene OsGSL1 was up‐regulated more and that the callose hydrolase (β‐1,3‐glucanase) gene Gns5 was up‐regulated less in Si‐amended than in nonamended plants during the initial stages of BPH infestation. These dynamic expression levels of OsGSL1 and Gns5 in response to BPH infestation correspond to callose deposition patterns in Si‐amended versus nonamended plants. It is demonstrated here that BPH infestation triggers differential gene expression associated with callose synthesis and hydrolysis in Si‐amended and nonamended rice plants, which allows callose to be deposited more on sieve tubes and sieve tube occlusions to be maintained more thus contributing to reduced BPH feeding on Si‐amended plants.     

OBSERVATIONS ON PENETRATION OF LINDEN BRANCHES BY STYLETS OF THE APHID LONGISTIGMA CARYAE

Penetration of the bark of Tilia americana L., the linden tree, by Longistigma caryae (Harr.) is mainly intracellular. Like other aphids, L. caryae secretes a saliva sheath which encloses the path of the stylets, beginning with an external collar of sheath material on the surface of the periderm. Stylet sheaths within the bark gave positive reactions for callose, suggesting that, in reaction to wounding, punctured parenchyma cells secrete callose which diffuses throughout the stylet sheaths. Other, more conspicuous effects of wounding included: proliferation and enlargement of cells of the cortex and dilated rays bordering some stylet sheaths, formation of tylosoids in punctured sieve elements, deposition of massive amounts of callose in penetrated sieve elements and in sieve elements bordering penetrated cells, and stimulation of cambial activity and xylem differentiation. Stylet tips located in living sieve elements projected beyond their sheaths which terminated outside the sieve-element walls. It is suggested that such sieve elements can be considered to be functional. None of the living sieve elements containing stylet tips showed any signs of injury which could be attributed to the presence of the stylets. Stylet tips of feeding aphids were found in living sieve elements of both 1965 and 1966 phloem increments clearly indicating that L. caryae can feed on linden sieve elements more than 1 year of age.     

Sieve-plate pores in leaf veins of Hordeum vulgare

Summary The sieve-plate pores of sieve elements in leaf veins of Hordeum vulgare, fixed in glutaraldehyde with postfixation in osmium tetroxide, were lined by the plasmalemma and variable amounts of callose. All pores were filled with endoplasmic reticulum, which was continuous from cell to cell. Mature sieve elements lacked P-protein.     

ANATOMICAL FEATURES OF METAPHLOEM IN STEMS OF SABAL,COCOS AND TWO OTHER PALMS

Sieve tubes in metaphloem of palm stems function throughout the life of the plant and merit close investigation. A stem of Sabal palmetto estimated to be 50 years old was sampled extensively. Variation in length of sieve-tube elements throughout this stem was measured and is discussed. In the metaphloem of individual vascular bundles companion cells are not sharply differentiated from other phloem parenchyma cells. Definitive callose deposits and slime are normally absent from mature sieve tubes, even in fixed material. Otherwise no conspicuous structural features which might account for the longevity of sieve tubes can be discerned. Occlusion of phloem strands after leaf fall is initially by callose deposition on sieve plates followed immediately by tylosoid formation. Similar sampling of Cocos nucifera, Washingtonia robusta and to a lesser extent Archontophoenix alexandrae confirmed these results except for quantitative differences.     

Role of auxin and sucrose in the differentiation of sieve and tracheary elements in plant tissue cultures

The differentiation of sieve and tracheary elements was studied in callus culture of Daucus carota L., Syringa vulgaris L., Glycine max (L.) Merr., Helianthus annuus L., Hibiscus cannabinus L. and Pisum sativum L. By the lacmoid clearing technique it was found that development of the phloem commenced before that of the xylem. In not one of the calluses was differentiation of tracheary elements observed in the absence of sieve elements. The influence of indole-3-acetic acid (IAA) and sucrose was evaluated quantitatively in callus of Syringa, Daucus and Glycine. Low IAA levels resulted in the differentiation of sieve elements with no tracheary cells. High levels resulted in that of both phloem and xylem. IAA thus controlled the number of sieve and tracheary elements, increase in auxin concentration boosting the number of both cell types. Changes in sucrose concentration, while the IAA concentration was kept constant, did not have a specific effect on either sieve element differentiation, or on the ratio between phloem and xylem. Sucrose did, however, affect the quantity of callose deposited on the sieve plates, because increase in the sucrose concentration resulted in an increase in the amount of callose. It is proposed that phloem is formed in response to auxin, while xylem is formed in response to auxin together with some added factor which reaches it from the phloem.     

Callose deposition in the phloem plasmodesmata and inhibition of phloem transport in citrus leaves infected with “Candidatus Liberibacter asiaticus”

Huanglongbing (HLB) is a destructive disease of citrus trees caused by phloem-limited bacteria, Candidatus Liberibacter spp. One of the early microscopic manifestations of HLB is excessive starch accumulation in leaf chloroplasts. We hypothesize that the causative bacteria in the phloem may intervene photoassimilate export, causing the starch to over-accumulate. We examined citrus leaf phloem cells by microscopy methods to characterize plant responses to Liberibacter infection and the contribution of these responses to the pathogenicity of HLB. Plasmodesmata pore units (PPUs) connecting companion cells and sieve elements were stained with a callose-specific dye in the Liberibacter-infected leaf phloem cells; callose accumulated around PPUs before starch began to accumulate in the chloroplasts. When examined by transmission electron microscopy, PPUs with abnormally large callose deposits were more abundant in the Liberibacter-infected samples than in the uninfected samples. We demonstrated an impairment of symplastic dye movement into the vascular tissue and delayed photoassimilate export in the Liberibacter-infected leaves. Liberibacter infection was also linked to callose deposition in the sieve plates, which effectively reduced the sizes of sieve pores. Our results indicate that Liberibacter infection is accompanied by callose deposition in PPUs and sieve pores of the sieve tubes and suggest that the phloem plugging by callose inhibits phloem transport, contributing to the development of HLB symptoms.     

CalS7 encodes a callose synthase responsible for callose deposition in the phloem

It has been known for more than a century that sieve plates in the phloem in plants contain callose, a β-1,3-glucan. However, the genes responsible for callose deposition in this subcellular location have not been identified. In this paper we examine callose deposition patterns in T-DNA insertion mutants (cs7) of the Callose Synthase 7 (CalS7) gene. We demonstrated here that the CalS7 gene is expressed specifically in the phloem of vascular tissues. Callose deposition in the phloem, especially in the sieve elements, was greatly reduced in cs7 mutants. Ultrastructural analysis of developing sieve elements revealed that callose failed to accumulate in the plasmodesmata of incipient sieve plates at the early perforation stage of phloem development, resulting in the formation of sieve plates with fewer pores. In wild-type Arabidopsis plants, callose is present as a constituent polysaccharide in the phloem of the stem, and its accumulation can also be induced by wounding. Callose accumulation in both conditions was eliminated in mature sieve plates of cs7 mutants. These results demonstrate that CalS7 is a phloem-specific callose synthase gene, and is responsible for callose deposition in developing sieve elements during phloem formation and in mature phloem induced by wounding. The mutant plants exhibited moderate reduction in seedling height and produced aberrant pollen grains and short siliques with aborted embryos, suggesting that CalS7 also plays a role in plant growth and reproduction.     

OBSERVATIONS ON SIEVE ELEMENTS IN THREE PERENNIAL MONOCOTYLEDONS

Seasonal collections were made of rhizomes of Polygonatum canaliculatum and Typha latifolia and of aerial stems of Smilax hispida. Many metaphloem sieve elements in all three species remain functional for 2 or more years, or for the life of the plant parts in which they occur. Although the protoplasts of mature sieve elements remain similar in appearance from one time of year to the next, the amount of callose associated with the sieve plates and lateral sieve areas of such cells apparently varies with the seasons, being heavier in late fall and winter and lighter in late spring and summer. At maturity the metaphloem sieve elements contain strands derived from the slime bodies of immature cells. It is suggested that in mature sieve elements the slime strands normally occur as a network along the wall. Many mature sieve elements of S. hispida contained normal-appearing nuclei.     

Ultrastructure of minor veins inCucurbita pepo leaves

Summary The minor veins ofCucurbita pepo leaves were examined as part of a continuing study of leaf development and phloem transport in this species. The minor veins are bicollateral along their entire length. Mature sieve elements are enucleate and lack ribosomes. There is no tonoplast. The sieve elements, which are joined to each other by sieve plates, contain mitochondria, plastids and endoplasmic reticulum as well as fibrillar and tubular (190–195 diameter) P-protein. Fibrillar P-protein is dispersed in mature abaxial sieve elements but remains aggregated as discrete bodies in mature adaxial sieve elements. In both abaxial and adaxial mature sieve elements tubular P-protein remains undispersed. Sieve pores in abaxial sieve elements are narrow, lined with callose and are filled with P-protein. In adaxial sieve elements they are wide, contain little callose and are unobstructed. The intermediary cells (companion cells) of the abaxial phloem are large and dwarf the diminutive sieve elements. Intermediary cells are densely filled with ribosomes and contain numerous small vacuoles and many mitochondria which lie close to the plasmalemma. An unusually large number of plasmodesmata traverse the common wall between intermediary cells and bundle sheath cells suggesting that the pathway for the transport of photosynthate from the mesophyll to the sieve elements is at least partially symplastic. Adaxial companion cells are of approximately the same diameter as the adaxial sieve elements. They are densely packed with ribosomes and have a large central vacuole. They are not conspicuously connected by plasmodesmata to the bundle sheath.     

Occurrence of Anomalous Mycoplasma-like Organisms in Grapevine Yellows-diseased Phloem

Pathological changes in Vitis vinifera cv. Caveccia phloem from leaves showing symptoms of a flavescence doreé-like disease consisted of obliteration, necrosis and collapse of the sieve elements and associated companion cells, and excessive callose accumulation in lateral sieve areas and sieve plates of apparently normal mature sieve elements. Unusual structures, also found in degenerate sieve elements of diseased leaf vein specimens, were strongly electron-dense and bounded by a unit membrane or an electron-transparent border, and considered to be senescent forms of mycoplasma-like organisms. The significance of these findings in relation to possible host responses to the yellows pathogen is discussed.     

Occurrence of Anomalous Mycoplasma-like Organisms in Grapevine Yellows-diseased Phloem

Pathological changes in Vitis vinifera cv. Caveccia phloem from leaves showing symptoms of a flavescence doreé-like disease consisted of obliteration, necrosis and collapse of the sieve elements and associated companion cells, and excessive callose accumulation in lateral sieve areas and sieve plates of apparently normal mature sieve elements. Unusual structures, also found in degenerate sieve elements of diseased leaf vein specimens, were strongly electron-dense and bounded by a unit membrane or an electron-transparent border, and considered to be senescent forms of mycoplasma-like organisms. The significance of these findings in relation to possible host responses to the yellows pathogen is discussed.     

Phloem Translocation and Heat-induced Callose Formation in Field-grown Gossypium hirsutum L

Phloem translocation rates in field-grown cotton (Gossypium hirsutum L.) dropped from morning to afternoon and continued to decline toward evening, except that recovery occurred following the hottest afternoon when the maximum temperature was 44 C. Water deficits increased from morning to evening, and severity of deficits generally were proportional to daytime heating. Water stress contributed toward reducing translocation but was not always the governing factor. Callose breakdown appeared to be slower than heat-induced synthesis, and in the evening callose still reflected the influence of high afternoon temperatures. Translocation was considerably reduced when about 50% or more of the hypocotyl sieve plates had large amounts of callose. While heat-induced callose may have reduced translocation because of sieve plate pore constriction, temperatures of 39 to 44 C appeared to inhibit an additional component of translocation as well, possibly in the leaf blade.     

STRUCTURAL CHANGES DURING BEAN LEAF ABSCISSION

Anatomical changes in the laminar abscission zone of primary leaves of Phaseolus vulgaris L. ‘Red Kidney’ have been examined in conjunction with the regulation of abscission by growth substances. Quantitative measurements were made of the frequency of vascular obstructions (tyloses, callose plugs). The development of abscission was correlated with an increasing frequency of tyloses and other plugging materials in the xylem of the abscission zone coupled with the dissolution of callose from the abscission zone sieve tubes. These changes were accelerated in petiole explants in which abscission was stimulated by either ethylene or auxin and were suppressed in explants in which abscission was inhibited by auxin.     

Über die Bildung von Kallose bei einer Hemmung des Transportes in den Siebröhren durch Cyanid

Zusammenfassung Durch vergleichende Untersuchung der Transporthemmung und der Kallosebildung in unbehandelten und cyanidvergifteten Zentralleitbündeln der Blattstiele vonPelargonium zonale wurde die Frage einer mechanischen Transporthemmung durch die Kallose geprüft. Cyanid regte in der verwendeten Konzentration die Kallosebildung bei einem Teil der Siebplatten deutlich an; eine Rückbildung in Leitbündeln, die nach einer Erholungszeit wieder Fluorescein leiteten, wurde nicht beobachtet, doch nahm mit dem Alter der Blattstiele die Kallosemenge auch ohne Vergiftung erheblich zu. Anhand der Ergebnisse wird eine rein mechanische Transporthemmung durch Cyanid infolge von Kallosebildung für unwahrscheinlich gehalten.

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Summary The problem of a mechanical inhibition of transport by callose formation was tested in comparative studies of the inhibition of transport and the formation of callose with untreated and cyanide-treated central bundles of the leaf petioles ofPelargonium zonale. Cyanide, in the applied concentration, clearly stimulated callose formation on many sieve plates. A disappearance of callose in bundles which, after a time of recovery, were able to transport again was not observed, but, even without poisoning, callose formation still increased markedly during the aging of the petioles. A mechanical inhibition of transport by cyanide-induced callose formation is, therefore, thought to be unlikely.

     

Ca2+-mediated remote control of reversible sieve tube occlusion in Vicia faba

According to an established concept, injury of the phloem triggers local sieve plate occlusion including callose-mediated constriction and, possibly, protein plugging of the sieve pores. Sieve plate occlusion can also be achieved by distant stimuli, depends on the passage of electropotential waves (EPWs), and is reversible in intact plants. The time-course of the wound response was studied in sieve elements of main veins of intact Vicia faba plants using confocal and multiphoton microscopy. Only 15-45 s after burning a leaf tip, forisomes (giant protein bodies specific for legume sieve tubes) suddenly dispersed, as observed at 3-4 cm from the stimulus site. The dispersion was reversible; the forisomes had fully re-contracted 7-15 min after burning. Meanwhile, callose appeared at the sieve pores in response to the heat shock. Callose production reached a maximum after approximately 20 min and was also reversible; callose degraded over the subsequent 1-2 h. The heat induction of both modes of occlusion coincided with the passage of an EPW visualized by electrophysiology or the potential-sensitive dye RH-414. In contrast to burning, cutting of the leaf tip induced neither an EPW nor callose deposition. The data are consistent with a remote-controlled occlusion of sieve plates depending on the longitudinal propagation of an EPW releasing Ca(2+) into the sieve element lumen. It is hypothesized that forisome plugs and callose constriction are removed once the cytosolic calcium level has returned to the initial level in those sieve tubes.     

SOME FINE STRUCTURAL OBSERVATIONS ON DEVELOPING AND MATURE SIEVE ELEMENTS IN THE BROWN ALGA LAMINARIA SACCHARINA

The differentiation of sieve elements from inner cortical cells of the stipe of Laminaria saccharina (L.) Lamour. involves the development of a well-structured protoplast and an end wall possessing evenly spaced pores which are visualized by electron microscopy. The protoplast consists of organelles which are commonly found in brown algal cells, including nuclei, cup- or horseshoe-shaped chloroplasts, dictyosomes, mitochondria, and ER. Mitochondria and clusters of small vacuoles, presumably redistributed by the surging effect which occurs in sieve elements, were routinely observed in the vicinity of the end wall. Chloroplasts were seen in progressively degenerated states in older sieve elements, yet nuclei were determined to be non-necrotic. Numerous pores along the end walls interconnect adjacent sieve elements. Each pore is traversed by a strand of cytoplasm and surrounded by plasmalemma. The pores are open and possess no callose. In this paper the sieve element ultrastructures of L. saccharina are compared to those in L. groenlandica, Alaria marginata, Nereocystis lutkeana and Macrocystis pyrifera, and a possible phylogenetic specialization of sieve elements is presented in table form and discussed.