Plant- and stimulus-specific variations in remote-controlled sieve-tube occlusion

Phloem injury triggers local sieve-plate occlusion including callose-mediated constriction and protein plugging of sieve pores. In intact plants, reversible sieve-plate occlusion is induced by electric potential waves (EPWs)—accompanied by Ca²⁺-influx—as result of distant burning. Here, we present additional results which pertain to (a) the variability of EPW-profiles in relation to forisome conformation in intact Vicia faba plants and (b) the differential occlusion reactions to burning and cutting in various plant species. A correlation between stimulus perception and mode of phloem loading is discussed.Key words: callose, electrical potential waves, forisome, membrane potential, phloem transport, sieve-element occlusion, wound potentials     

The geometry of the forisome-sieve element-sieve plate complex in the phloem of Vicia faba L. leaflets

Forisomes are contractile protein bodies that appear to control flux rates in the phloem of faboid legumes by reversibly plugging the sieve tubes. Plugging is triggered by Ca(2+) which induces an anisotropic deformation of forisomes, consisting of a longitudinal contraction and a radial expansion. By conventional light microscopy and confocal laser-scanning microscopy, the three-dimensional geometry of the forisome-sieve element-sieve plate complex in intact sieve tubes of leaflets of Vicia faba L. was reconstructed. Forisomes were mostly located close to sieve plates, and occasionally were observed drifting unrestrainedly along the sieve element, suggesting that they might be utilized as internal markers of flow direction. The diameter of forisomes in the resting state correlated with the diameter of their sieve elements, supporting the idea that radial expansion of forisomes is the geometric basis of reversible sieve tube plugging. Comparison of the present results regarding forisome geometry in situ with previously published data on forisome reactivity in vitro makes it questionable, however, whether forisomes are capable of completely sealing sieve tubes in V. faba leaves.     

Similar Intracellular Location and Stimulus Reactivity,but Differential Mobility of Tailless (Vicia faba) and Tailed Forisomes (Phaseolus vulgaris) in Intact Sieve Tubes

Sieve elements of legumes contain forisomes—fusiform protein bodies that are responsible for sieve-tube occlusion in response to damage or wound signals. Earlier work described the existence of tailless and tailed forisomes. This study intended to quantify and compare location and position of tailless (in Vicia faba) and tailed (in Phaseolus vulgaris) forisomes inside sieve elements and to assess their reactivity and potential mobility in response to a remote stimulus. Location (distribution within sieve elements) and position (forisome tip contacts) of more than altogether 2000 forisomes were screened in 500 intact plants by laser scanning confocal microscopy in the transmission mode. Furthermore, we studied the dispersion of forisomes at different locations in different positions and their positional behaviour in response to distant heat shocks. Forisome distribution turned out to be species-specific, whereas forisome positions at various locations were largely similar in bushbean (Phaseolus) and broadbean (Vicia). In general, the tailless forisomes had higher dispersion rates in response to heat shocks than the tailed forisomes and forisomes at the downstream (basal) end dispersed more frequently than those at the upstream end (apical). In contrast to the tailless forisomes that only oscillate in response to heat shocks, downstream-located tailed forisomes can cover considerable distances within sieve elements. This displacement was prevented by gentle rubbing of the leaf (priming) before the heat shock. Movement of these forisomes was also prohibited by Latrunculin A, an inhibitor of actin polymerization. The apparently active mobility of tailed forisomes gives credence to the idea that at least the latter forisomes are not free-floating, but connected to other sieve-element structures.     

Forisome performance in artificial sieve tubes

In the legume phloem, sieve element occlusion (SEO) proteins assemble into Ca(2+)-dependent contractile bodies. These forisomes presumably control phloem transport by forming reversible sieve tube plugs. This function, however, has never been directly demonstrated, and appears questionable as forisomes were reported to be too small to plug sieve tubes, and failed to block flow efficiently in artificial microchannels. Moreover, plugs of SEO-related proteins in Arabidopsis sieve tubes do not affect phloem translocation. We improved existing procedures for forisome isolation and storage, and found that the degree of Ca(2+)-driven deformation that is possible in forisomes of Vicia faba, the standard object of earlier research, has been underestimated substantially. Forisomes deform particularly strongly under reducing conditions and high sugar concentrations, as typically found in sieve tubes. In contrast to our previous inference, Ca(2+)-inducible forisome swelling certainly seems sufficient to plug sieve tubes. This conclusion was supported by 3D-reconstructions of forisome plugs in Canavalia gladiata. For a direct test, we built microfluidics chips with artificial sieve tubes. Using fluorescent dyes to visualize flow, we demonstrated the complete blockage of these biomimetic microtubes by Ca(2+)-induced forisome plugs, and concluded by analogy that forisomes are capable of regulating phloem flow in vivo.     

Translocation blockage by sieve plate callose

Summary Axial translocation in 2-week-old cotton plants was inhibited by heating 4 cm of intact hypocotyl for 15 min by means of a 40–45° water jacket. A 1-cm jacket did not retard translocation, and temperatures below 40° had no effect. Translocation continued to be inhibited for at least 3 hours following heat treatment. After 6 hours, rates were equal to or above normal. Maximum amounts of callose were deposited on sieve plates after the heat treatment, but callose was noticeably diminished within 6 hours after heating and reduced to virtually normal levels within 2 days. Growth measurements, plasmolytic tests, vital staining, and visual observations revealed no evidence of injury in plants heated at 45°. Pore constriction from increased amounts of callose on sieve plates appears to be an effect of heating. Increased resistance due to such constriction may be an important factor in blockage of basipetal phloem translocation.Work supported in part by National Science Foundation Grant GB-2941. This material is abstracted from a dissertation presented in 1967 by R. B. McNairn to the Graduate Division, University of California at Davis in partial fulfillment of the requirements for the Ph. D. degree.All temperatures in this paper are in degrees centigrade (°C)     

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.     

Sieve Element Ca2+ Channels as Relay Stations between Remote Stimuli and Sieve Tube Occlusion in Vicia faba

Damage induces remote occlusion of sieve tubes in Vicia faba by forisome dispersion, triggered during the passage of an electropotential wave (EPW). This study addresses the role of Ca²⁺ channels and cytosolic Ca²⁺ elevation as a link between EPWs and forisome dispersion. Ca²⁺ channel antagonists affect the initial phase of the EPW as well as the prolonged plateau phase. Resting levels of sieve tube Ca²⁺ of ∼50 nM were independently estimated using Ca²⁺-selective electrodes and a Ca²⁺-sensitive dye. Transient changes in cytosolic Ca²⁺ were observed in phloem tissue in response to remote stimuli and showed profiles similar to those of EPWs. The measured elevation of Ca²⁺ in sieve tubes was below the threshold necessary for forisome dispersion. Therefore, forisomes need to be associated with Ca²⁺ release sites. We found an association between forisomes and endoplasmic reticulum (ER) at sieve plates and pore-plasmodesma units where high-affinity binding of a fluorescent Ca²⁺ channel blocker mapped an increased density of Ca²⁺ channels. In conclusion, propagation of EPWs in response to remote stimuli is linked to forisome dispersion through transiently high levels of parietal Ca²⁺, release of which depends on both plasma membrane and ER Ca²⁺ channels.     

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     

(Questions) on phloem biology. 1. Electropotential waves, Ca fluxes and cellular cascades along the propagation pathway

This review explores the relationships between electrical long-distance signalling, Ca²⁺ influx coincident with propagation of electropotential waves, and cellular responses to Ca²⁺ influx including the consequences for sieve-tube conductivity and mass flow. Ca²⁺ influx is inherent to electropotential waves and appears to constitute the key link between rapid physical signals and resultant chemical cascades in sieve tubes and adjacent cells. Members of several channel groups are likely involved the regulation of Ca²⁺ levels in sieve elements. Among them are hyperpolarization-activated, depolarization-activated, and mechanosensitive Ca²⁺ channels located in the plasma membrane and Ca²⁺ dependent Ca²⁺ channels that reside in ER-membranes of sieve elements. These channels collectively determine intracellular Ca²⁺ levels in sieve elements and their neighbour cells. The latter cells react to Ca²⁺ elevation by inducing diverse functional responses dependent on the cell type. If the Ca²⁺ concentration in sieve elements surpasses a threshold level, dual sieve-plate occlusion by proteins and callose deposition is triggered. Occlusion is reversed when Ca²⁺ levels subside. Electrical messages may regulate the degree of sieve plate hydraulic conductivity in intact plants by partial sieve-plate occlusion that has a major impact on volume flow through sieve tubes. Furthermore, complete but temporary occlusion of sieve tubes may modify mass flow patterns in intact plants.     

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.     

Native and artificial forisomes: functions and applications

Forisomes are remarkable protein bodies found exclusively in the phloem of the Fabaceae. When the phloem is wounded, forisomes are converted from a condensed to a dispersed state in an ATP-independent reaction triggered by Ca²⁺, thereby plugging the sieve tubes and preventing the loss of photoassimilates. Potentially, forisomes are ideal biomaterials for technical devices because the conformational changes can be replicated in vitro and are fully reversible over a large number of cycles. However, the development of technical devices based on forisomes has been hampered by the laborious and time-consuming process of purifying native forisomes from plants. More recently, the problem has been overcome by the production of recombinant artificial forisomes. This is a milestone in the development of forisome-based devices, not only because large quantities of homogeneous forisomes can be produced on demand, but also because their properties can be tailored for particular applications. In this review, we discuss the physical and molecular properties of native and artificial forisomes, focusing on their current applications in technical devices and potential developments in the future.     

Characteristics of artificial forisomes from plants and yeast

Forisomes are protein bodies found exclusively in the phloem of the Fabaceae (legumes). In response to wounding, the influx of Ca ( 2+) induces a conformational change from a condensed to a dispersed state which plugs the sieve tubes and prevents the loss of photoassimilates. This reversible, ATP-independent reaction can be replicated with purified forisomes in vitro by adding divalent cations or electrically inducing changes in pH, making forisomes ideal components of technical devices. Although native forisomes comprise several subunits, we recently showed that functional homomeric forisomes with distinct properties can be expressed in plants and yeast, providing an abundant supply of forisomes with tailored properties. Forisome subunits MtSEO-F1 and MtSEO-F4 can each assemble into homomeric artificial forisomes, which indicates functional redundancy. However, we provide further evidence that both proteins are subunits of the native heteromeric forisome body in planta. We also show that the properties of artificial forisomes can be modified by immobilization, which is a prerequisite for their incorporation into technical devices.     

Tailed forisomes of Canavalia gladiata: a new model to study Ca2+-driven protein contractility

BACKGROUND AND AIMS: Forisomes are Ca(2+)-dependent contractile protein bodies that form reversible plugs in sieve tubes of faboid legumes. Previous work employed Vicia faba forisomes, a not entirely unproblematic experimental system. The aim of this study was to seek to establish a superior model to study these intriguing actuators. METHODS: Existing isolation procedures were modified to study the exceptionally large, tailed forisomes of Canavalia gladiata by differential interference contrast microscopy in vitro. To analyse contraction/expansion kinetics quantitatively, a geometric model was devised which enabled the computation of time-courses of derived parameters such as forisome volume from simple parameters readily determined on micrographs. KEY RESULTS: Advantages of C. gladiata over previously utilized species include the enormous size of its forisomes (up to 55 microm long), the presence of tails which facilitate micromanipulation of individual forisomes, and the possibility of collecting material repeatedly from these fast-growing vines without sacrificing the plants. The main bodies of isolated Canavalia forisomes were box-shaped with square cross-sections and basically retained this shape in all stages of contraction. Ca(2+)-induced a 6-fold volume increase within about 10-15 s; the reverse reaction following Ca(2+)-depletion proceeded in a fraction of that time. CONCLUSIONS: The sword bean C. gladiata provides a superior experimental system which will prove indispensable in physiological, biophysical, ultrastructural and molecular studies on the unique ATP-independent contractility of forisomes.     

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.     

Preservation of the Tonoplast in Metaphloem Sieve Elements of Embryonic Roots of Zea mays L.

Decortication of embryonic roots of 4- to 5-day-old Zea seedlingsand subsequent chemical fixation permitted comparison of cutand uncut developing sieve elements In a decorticated root wheresieve tubes are not severed, metaphloem sieve elements in latestates of development and some mature sieve elements exhibita highly vacuolate condition When roots are cut or diced inthe course of fixation intact vacuoles are not observed in latestages of sieve-element ontogeny The degree of callose formationat sites of developing sieve-plate pores and in the pores ofmature sieve elements varies greatly with both decorticationand non-decortication treatments Nuclei were not observed insieve elements at the electron microscope level, but they wereseen at the light microscope level in serial sections of sieveelements in the late to mature developmental stages representedAlthough the occurrence and distribution of plastids, mitochondria,endoplasmic reticulum, dictyosomes and nbosomes also vanes insieve elements of decorticated roots, disruption or surgingof sieve-element contents is greater for sieve tubes that aresevered during fixation treatment A discussion is presentedrelating effects of trauma on observed developmental stagesand sieve-element structure Zea mays L, maize, corn, phloem, Sieve elements, tonoplast, ultrastructure     

Calcium-energized motor protein forisome controls damage in phloem: potential applications as biomimetic “smart” material

Forisomes are ATP independent, mechanically active proteins from the Fabaceae family (also called Leguminosae). These proteins are located in sieve tubes of phloem and function to prevent loss of nutrient-rich photoassimilates, upon mechanical injury/wounding. Forisomes are SEO (sieve element occlusion) gene family proteins that have recently been shown to be involved in wound sealing mechanism. Recent findings suggest that forisomes could act as an ideal model to study self assembly mechanism for the development of nanotechnological devices like microinstruments, the microfluidic system frequently used in space exploration missions. Technology enabling improvement in micro instruments has been identified as a key technology by NASA in future space exploration missions. Forisomes are designated as biomimetic smart materials which are calcium-energized motor proteins. Since forisomes are biomolecules from plant systems it can be doctored through genetic engineering. In contrast, “smart” materials which are not derived from plants are difficult to modify in their properties. Current levels of understanding about forisomes conformational shifts with respect to calcium ions and pH changes requires supplement of future advances with relation to its 3D structure to understand self assembly processes. In plant systems it forms blood clots in the form of occlusions to prevent nutrient fluid leakage and thus proves to be a unique damage control system of phloem tissue.     

SIEVE TUBES AND CALLOSE IN ELODEA LEAVES

Detachment and incubation of Elodea leaves promoted callose synthesis in all cells, especially in epidermal pits and in sieve tubes. Phloem was detected in the midrib by fluorescent staining of callose induced to form on sieve plates. In EM views of mature sieve elements nucleus and tonoplast were lacking, mictoplasm replaced cytoplasm, mitochondria were fewer in number, and large plastids contained crystalline inclusion bodies. Slime was present as compact aggregates and as individual fibrils in mictoplasm and sieve pores. Deposition of callose is considered in relation to the blockage concept of callose function.     

THE FINE STRUCTURE OF SIEVE ELEMENTS OF NEREOCYSTIS LÜTKEANA

The sieve elements of Nereocystis from the base of phylloids contain numerous small vesicles, cytoplasm, ribosomes, and the usual organelles and membrane systems, including nuclei, plastids, mitochondria, dictyosomes, and endoplasmic reticulum. They have a thick secondary wall layer which is deposited along the longitudinal walls and at the sieve plate excluding the sieve pores. The sieve pores range in diameter from 100 to 400 nm and are lined by plasmalemma. The sieve elements from the hollow basal parts of the pneumatocyst show essentially the same features but have larger and fewer vesicles, relatively little cytoplasm, larger sieve pores, 400–900 nm in diameter, and may lack a nucleus. In old sieve elements there are large deposits of callose on the sieve plate and along the longitudinal wall; the vesicles seem to break down, and the protoplast appears necrotic. It is concluded that the trumpet hyphae and sieve tubes are basically the same type of cell, and that the trumpet-shape of the sieve elements is due to their passive stretching during extension growth of the organ in which they occur. There are minor but significant differences among the sieve elements from different regions of the thallus which may reflect possible levels of structural specialization of the sieve elements within the same plant.