Sieve Element of Impatiens sultanii: 1. Wound Reaction

A Study of wound reaction of the metaphloem of the stem by white-light,fluorescence, and electron microscopy provides evidence forthe structure of mature sieve elements in the intact plant.Starch grains usually are retained in plastids which are locatedagainst the lateral walls of sieve elements and concentratedat both ends of each cell. Slime plugs and dense connectingstrands in the sieve plates seem to result from reactions tocutting or penetration of the killing agent; after appropriatemethods of killing, the contents of a connecting strand maybe only slightly denser, if any, than the milieu on either sideof the sieve plate. A strange accumulation of slime, consistingof streamers directed toward the wound surface from each sieveplate, occurred in tissue boiled immediately after incisionof the phloem. Callose is present in sieve elements of intactplants when the tissue is killed within 4 seconds after injury.Callose is accumulated in response to wounding in added amounts,but only after 5 minutes or more and only within about 15 sieveelements from the wound. Quantities of callose sufficient forplugging the sieve plate accumulate after 30 minutes or more.Sieve-plate callose and deposits on the nearby lateral wallsproduce a cup-shaped mass which is called a cup deposit.     

A Rapid Method for Macerating Phloem

Sieve cells and sieve tube members can be macerated from the phloem of various organs of woody and herbaceous species by au-toclaving the tissue in a mild macerating medium. This treatment does not digest the primary walls or the callose deposits on the sieve areas and sieve plates of the sieve elements. These cells can then be recognized by the fluorescence of their callose after staining with aniline blue. Sometimes adjacent sieve elements fail to separate and one can observe details of their junctures.     

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.     

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.     

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.     

SOME ULTRASTRUCTURAL ASPECTS OF METAPHLOEM SIEVE ELEMENTS IN THE AERIAL STEM OF THE HOLOPARASITIC ANGIOSPERM EPIFAGUS VIRGINIANA (OROBANCHACEAE)

A light and electron microscope investigation was conducted on phloem in the aerial stem of Epifagus virginiana (L.) Bart. Tissue was processed at field collection sites in an effort to overcome problems resulting from manipulation. At variance with earlier accounts, Epifagus phloem consists of sieve elements, companion cells, phloem parenchyma cells, and primary phloem fibers. The sieve elements possess simple sieve plates and the phloem is arranged in a collateral type of vascular bundle. In addition, this constitutes the first study on phloem ultrastructure in the aerial stems of a holoparasitic dicotyledon, an entire plant which could be viewed as an “ideal sink.” Epifagus phloem possesses unoccluded sieve plate pores in mature sieve elements and a total lack of P-protein in sieve elements at all stages of development. Mature sieve elements lack nuclei. Plastids were rarely observed in mature sieve elements. Vacuoles with intact tonoplasts were encountered in some mature sieve elements. Otherwise, the ultrastructural features of sieve elements appear to differ little from those described by investigators of non-parasitic species.     

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.     

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.     

OBSERVATIONS ON METAPHLOEM IN THE VEGETATIVE PARTS OF PALMS

Metaphloem was studied in available vegetative parts of 374 species in 164 genera of palms. Sieve elements usually have compound sieve plates except in the subfamilies Lepidocaryoideae and Nypoideae. Sieve elements in roots usually have oblique to very oblique end walls, whereas in stems and leaves they have transverse to oblique walls. Within a phloem strand the degree of compounding of a sieve plate is directly correlated with element diameter. Plastids are normally present in functioning, enucleate sieve elements. Small quantities of “slime” substances have been detected in young sieve elements in stems and petioles of a few species. Many sieve plates in functioning sieve elements lacked callose in materials quick-killed in liquid nitrogen or chilled acetic-alcohol. Definitive callose is confined to sieve elements just before their obliteration. Sieve tubes in leaf and stem are usually ensheathed by contiguous parenchyma cells while those in root have very few contiguous parenchyma cells. Two types of contiguous parenchyma cells can be distinguished by difference in cytoplasmic density, especially with the electron microscope. Cells with denser cytoplasm are interpreted as companion cells. Lignified contiguous parenchyma cells are occasionally present in metaphloem of petioles. The possible diagnostic and taxonomic features of metaphloem are discussed.     

Stem anatomy and development of inter- and intraxylary phloem in Leptadenia pyrotechnica (Forssk.) Decne. (Asclepiadaceae)

Modification of external morphology and internal structure of plants is a key feature of their successful survival in extreme habitats. They adapt to arid habitats not only by modifying their leaves, but also show several modifications in their conducting system. Therefore, the present study is aimed to investigate the pattern of secondary growth in Leptadenia pyrotechnica (Forssk.) Decne., (Asclepiadaceae), one such species growing in Kachchh district, an arid region of Gujarat State. A single ring of vascular cambium, responsible for radial growth, divided bidirectionally and formed the secondary xylem centripetally and the phloem centrifugally. After a short period of secondary xylem differentiation, small arcs of cambium began to form secondary phloem centripetally instead of secondary xylem. After a short duration of such secondary phloem formation, these segments of cambium resumed their normal function to produce secondary xylem internally. Thus, the phloem strands became embedded within the secondary xylem and formed interxylary phloem islands. Such a recurrent behavior of the vascular cambium resulted in the formation of several patches of interxylary phloem islands. In thick stems the earlier formed non-conducting interxylary phloem showed heavy accumulation of callose on the sieve plates followed by their crushing in response to the addition of new sieve elements. Development of intraxylary phloem is also observed from the cells situated on the pith margin. As secondary growth progresses further, small arcs of internal cambium get initiated between the protoxylem and intraxylary phloem. In the secondary xylem, some of the vessels are exceptionally thick-walled, which may be associated with dry habitats in order to protect the vessel from collapsing during the dryer part of the year. The inter- and intraxylary phloem may also be an adaptive feature to prevent the sieve elements to become non-conducting during summer when the temperature is much higher.     

SEASONAL DEVELOPMENT OF THE SECONDARY PHLOEM IN ACER NEGUNDO

Functional sieve elements are present year-round in the secondary phloem of the trunk of Acer negundo L., the box elder tree. Judging from numerous collections made between May, 1962, and May, 1964, the seasonal cycle of phloem development is as follows: cambial activity and new phloem differentiation begin in late March or early April; xylem differentiation begins about a month later and is completed in most trees in late August. At the time of cessation of cambial activity most of the relatively wide sieve elements of the current season's increment are mature. However, numerous groups of narrow, immature sieve elements and companion cells located on the outer margin of the cambial zone do not reach maturity until fall and winter. By the time of cambial reactivation in spring, most, if not all, of these narrow elements are mature. Some of the sieve elements which reach maturity either shortly after cessation of cambial activity or during dormancy become non-functional within 6 weeks after resumption of cambial activity in spring, while others remain functional until mid-August. For the phloem increment of a given year, cessation of function begins in September with the accumulation of definitive callose on the sieve plates of the first-formed sieve elements and spreads to all but the last-formed ones by the end of December.     

Seasonal Changes in the Structure of the Secondary Phloem of Grewia tiliaefolia, a Deciduous Tree from India

Structure of the secondary phloem of Grewia tillaefolia Roxb.was studied in samples of bark collected at monthly intervalsfrom forest populations of Gujarat in western India. The secondaryphloem in this species is vertically storied and the axial elementsoccur as alternate tangential bands of fibres and sieve elementsproduced in succession. On average, two to four bands of fibresand corresponding bands of sieve elements are produced in ayear. The sieve elements function for more than one season anddifferent phloem increments are separated by terminal zonesmade up of very narrow sieve elements which mature just beforeand immediately after the period of dormancy. The tree becomesleafless about eight to ten weeks preceding the spring equinox.Cambial activity, phloem differentiation and phloem functionare suspended during this period. Differentiation of phloembegins after bud break which occurs in April, and continuesuntil January, but most of the phloem is produced between Julyand September when the rainy season is well advanced. The widthof the conducting zone is maximal at the end of the period ofgrowth when the tree is in full leaf. Inactivation of sieveelements, apparently by callose plugging the sieve plates, beginswith leaf abscission. The sieve elements produced in the precedingseason, just before dormancy is imposed resume function in thefollowing growing season and the older elements die. Companioncells and axial parenchyma cells surrounding sieve elementsappear to have s significant role during senescence of the conductingelements. The development and activity of the secondary phloemseem to be related to other developmental phenomena occurringwithin the tree.     

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.     

THE EFFECT OF BORON DEFICIENCY ON CALLOSE FORMATION AND 14C TRANSLOCATION IN BEAN (PHASEOLUS VULGARIS L.) AND COTTON (GOSSYPIUM HIRSUTUM L.)

Callose accumulated in the tissues of boron deficient bean and cotton plants, the extent and distribution of which depended on the species. Sieve plates in the phloem of boron deficient bean were characterized by heavy plugs of callose, while the sieve plates of boron deficient cotton were essentially unaffected. Translocation of ¹⁴C was, however, drastically reduced in both plants. It is suggested that callose deposition in boron deficient plants is a secondary effect of cellular damage.     

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.     

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     

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.     

The Phloem of Nelumbo nucifera Gaertn

In common with other aquatic angiosperms, Nelumbo nucifera Gaertn.has a relatively strongly developed phloem tissue. The vascularsystem consists of discrete collateral bundles in which no cambiumdevelops and the phloem and xylem are separated by a narrowlayer of parenchyma cells. The phloem consists of sieve elements,companion cells, and phloem parenchyma cells. The sieve elementshave transverse end walls with simple sieve plates. The cellsattain considerable width in the late phloem (metaphloem). Thecompanion cells are in vertical strands. In the early phloem(protophloem) of large bundles the sieve tubes and companioncells are eventually obliterated. The parenchyma cells alsoform vertical strands which may contain tannin cells. Some parenchymacells and companion cells are binucleate. The sieve elementsshow ultrastructural features common for these cells in dicotyledons.At maturity, they lack nuclei, ribosomes, and tonoplasts, butretain a plasmalemma, mitochondria, and plastids. The latterare poorly differentiated and form starch. The endoplasmic reticulumis in part stacked, in part it forms a network next to the plasmalemma.The P-protein occurs in two forms. One consists of tubules notassembled in any specific type of array. The other, possiblycomposed of much extended tubules, is assembled in crystallineaggregates which are retained as such in mature cells. The sieveplate pores are lined with callose and plasmalemma. The lateralwalls are relatively thin and the nacreous layer varies in degreeof distinctness.     

ULTRASTRUCTURAL FEATURES OF DEVELOPING SIEVE ELEMENTS IN LEMNA MINOR L.—SIEVE PLATE AND LATERAL SIEVE AREAS

Both intact and cut duckweed plants were prepared for electron microscopy. Plants which are prepared intact do not exhibit callose formation during development of sieve-plate pores. Future pore sites can be recognized by the presence of median cavities that are unassociated with callose platelets. These cavities are first seen in the region of the compound middle lamella and are lined by a plasmalemma. As end walls thicken, the cavities increase in size until open pores of uniform width are formed. Mature sieve plates of intact-prepared plants are also devoid of callose. Fully opened pores are lined by a plasmalemma and are only traversed by an occasional tubule of endoplasmic reticulum. Plants which have been cut prior to fixation possess mature sieve plates containing callose. The pores of developing sieve plates in cut plants exhibit small amounts of callose. Except for the lack of callose, lateral wall connections between sieve elements and contiguous cells are similar in development and mature state to those reported for other species.