Rapid cooling triggers forisome dispersion just before phloem transport stops

Phloem transport stops transiently within dicot stems that are cooled rapidly, but the cause remains unknown. Now it is known that (1) rapid cooling depolarizes cell membranes giving a transient increase in cytoplasmic Ca²⁺, and (2) a rise of free calcium triggers dispersion of forisomes, which then occlude sieve elements (SEs) of fabacean plants. Therefore, we compared the effects of rapid chilling on SE electrophysiology, phloem transport and forisomes in Vicia faba. Forisomes dispersed after rapid cooling with a delay that was longer for slower cooling rates. Phloem transport stopped about 20 s after forisome dispersion, and then transport resumed and forisomes re‐condensed within similar time frames. Transport interruption and forisome dispersion showed parallel behaviour – a cooling rate‐dependent response, transience and desensitization. Chilling induced both a fast and a slow depolarization of SE membranes, the electrical signature suggesting strongly that the cause of forisome dispersion was the transient promotion of SE free calcium. This apparent block of SEs by dispersed forisomes may be assisted by other Ca²⁺‐dependent sealing proteins that are present in all dicots.     

Legume phylogeny and the evolution of a unique contractile apparatus that regulates phloem transport

Protein bodies called forisomes undergo Ca(2+)-dependent deformations to occlude sieve tubes reversibly, providing a unique regulatory mechanism of phloem transport. Because forisomes are known exclusively from the Papilionoideae (Leguminosae), the evolution of forisome function may have played a role in the rapid radiation of this huge taxon. The unexpected discovery of a papilionoid species lacking forisomes led us to evaluate a representative set of species covering 33 of the 36 legume tribes traditionally recognized. We found forisomes in Papilionoideae but not in Caesalpinioideae and Mimosoideae. Forisomes were absent from several species of the papilionoid tribe Galegeae. Forisomes with tail-like protrusions occurred less frequently than tailless ones; their distribution correlated with taxonomic units but not sharply enough to render forisome type a reliable criterion for classification. Thus, the distribution of forisome types appeared to reflect physiological variability in the pathways of forisome assembly rather than the evolution of forisome genes. On the other hand, Ca(2+)-dependent forisome deformation and sieve tube plugging occurred in Bobgunnia madagascariensis, a member of the swartzioid clade that presumably is the sister group of all other papilionoids, suggesting that forisomes and their unique mechanism of deformation are a synapomorphy of the Papilionoideae.     

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.     

Penetration of faba bean sieve elements by pea aphid does not trigger forisome dispersal

Immediately after their stylets penetrate a phloem sieve element, aphids inject saliva into the sieve element for approximately 30–60 s before they begin to ingest phloem sap. This salivation period is recorded as waveform E1 in electrical penetration graph (EPG) monitoring of aphid feeding behavior. It has been hypothesized that the function of this initial period of phloem salivation is to reverse or prevent plugging of the sieve element by one of the plant's phloem defenses: formation of P‐protein plugs or callose synthesis in the sieve pores that connect adjacent sieve elements. This hypothesis was tested using the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), and faba bean, Vicia faba L. (Fabaceae), as a model system, and the results do not support the hypothesis. In legumes, such as faba bean, P‐protein plugs in sieve elements are formed by dispersal of proteinaceous bodies called forisomes. Contrary to the hypothesis, the great majority of sieve element penetrations by pea aphid stylets do not trigger forisome dispersal. Thirteen sieve elements were cryofixed early in phloem phase before the aphids could complete their salivation period and the forisomes were not dispersed in any of the 13 samples. However, in these samples, the aphids completed on average a little over half of their normal E1 salivation period before they were cryofixed. Thus, it is possible that sieve element penetration triggered forisome dispersal in these samples but the abbreviated period of salivation was still sufficient to reverse dispersal. To rule out this possibility, 17 sieve elements were cryofixed during R‐pds, which are an EPG waveform associated with sieve element penetration but without the characteristic E1 salivation that occurs during phloem phase. In 16 of the 17 samples, the forisomes were not dispersed. Thus, faba bean sieve elements usually do not form P‐protein plugs in response to penetration by pea aphid stylets. Consequently, the characteristic E1 salivation that occurs at the start of each phloem phase does not seem to be necessary to prevent a plugging response because penetration of sieve elements during R‐pds does not trigger forisome dispersal despite the absence of E1 salivation. Furthermore, as P‐protein plugs do not normally form in response to sieve element penetration, E1 salivation that occurs at the start of each phloem phase is not a response to development of a P‐protein plug. Thus, the E1 salivation period at the beginning of the phloem phase appears to have function(s) unrelated to phloem sealing.     

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.     

GFP tagging of sieve element occlusion (SEO) proteins results in green fluorescent forisomes

Forisomes are Ca(2+)-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as 'FOR' proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca(2+) and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that 'FOR'-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca(2+) binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism.     

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.     

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.     

The sieve element occlusion gene family in dicotyledonous plants

Sieve element occlusion (SEO) genes encoding forisome subunits have been identified in Medicago truncatula and other legumes. Forisomes are structural phloem proteins uniquely found in Fabaceae sieve elements. They undergo a reversible conformational change after wounding, from a condensed to a dispersed state, thereby blocking sieve tube translocation and preventing the loss of photoassimilates. Recently, we identified SEO genes in several non-Fabaceae plants (lacking forisomes) and concluded that they most probably encode conventional non-forisome P-proteins. Molecular and phylogenetic analysis of the SEO gene family has identified domains that are characteristic for SEO proteins. Here, we extended our phylogenetic analysis by including additional SEO genes from several diverse species based on recently published genomic data. Our results strengthen the original assumption that SEO genes seem to be widespread in dicotyledonous angiosperms, and further underline the divergent evolution of SEO genes within the Fabaceae.Key words: forisome, P-protein, sieve element occlusion, phloem, wound sealing, gene family, Fabacea     

Spatial and temporal regulation of the forisome gene <Emphasis Type="Italic">for1</Emphasis> in the phloem during plant development

Forisomes are protein aggregates found uniquely in the sieve elements of Fabaceaen plants. Upon wounding they undergo a reversible, calcium-dependent conformational switch which enables them to act as cellular stopcocks. Forisomes begin to form in young sieve elements at an early stage of metaphloem differentiation. Genes encoding forisome components could therefore be useful as markers of early sieve element development. Here we present a comprehensive analysis of the developmental expression profile of for1, which encodes such a forisome component. The for1 gene is highly conserved among Fabaceaen species and appears to be unique to this phylogenetic lineage since no orthologous genes have been found in other plants, including Arabidopsis and rice. Even so, transgenic tobacco plants expressing reporter genes under the control of the for1 promoter display reporter activity exclusively in immature sieve elements. This suggests that the regulation of sieve element development is highly conserved even in plants where mature forisomes have not been detected. The promoter system could therefore provide a powerful tool for the detailed analysis of differentiation in metaphloem sieve elements in an unexpectedly broad range of plant species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Forisome dispersion in Vicia faba is triggered by Ca2+ hotspots created by concerted action of diverse Ca2+ channels in sieve elements

Remote-controlled Ca²⁺ influx, elicited by electropotential waves, triggers local signaling cascades in sieve elements and companion cells along the phloem of Vicia faba plants. The stimulus strength seems to be communicated by the rate and duration of Ca²⁺ influx into sieve elements (SEs). The cooperative recruitment of Ca²⁺ channels results in a graded response of forisome culminating in full sieve-tube occlusion. Several lines of evidence are integrated into a model that links the mode and strength of the electropotential waves (EPWs) with forisome dispersion, mediated by transiently enhanced levels of local Ca²⁺ release dependent on both plasma membrane and ER Ca²⁺ channels.Key words: distant injury, electropotential wave, remote sieve tube occlusion, activity of sieve element Ca²⁺ channels, signal cascades, Ca²⁺ hotspots     

Faba bean forisomes can function in defence against generalist aphids

Phloem sieve elements have shut‐off mechanisms that prevent loss of nutrient‐rich phloem sap when the phloem is damaged. Some phloem proteins such as the proteins that form forisomes in legume sieve elements are one such mechanism and in response to damage, they instantly form occlusions that stop the flow of sap. It has long been hypothesized that one function of phloem proteins is defence against phloem sap‐feeding insects such as aphids. This study provides the first experimental evidence that aphid feeding can induce phloem protein occlusion and that the aphid‐induced occlusions inhibit phloem sap ingestion. The great majority of phloem penetrations in Vicia faba by the generalist aphids Myzus persicae and Macrosiphum euphorbiae triggered forisome occlusion and the aphids eventually withdrew their stylets without ingesting phloem sap. This contrasts starkly with a previous study on the legume‐specialist aphid, Acyrthosiphon pisum, where penetration of faba bean sieve elements did not trigger forisome occlusion and the aphids readily ingested phloem sap. Next, forisome occlusion was demonstrated to be the cause of failed phloem ingestion attempts by M. persicae: when occlusion was inhibited by the calcium channel blocker lanthanum, M. persicae readily ingested faba bean phloem sap.     

Molecular and phylogenetic characterization of the sieve element occlusion gene family in <Emphasis Type="Italic">Fabaceae</Emphasis> and non-<Emphasis Type="Italic">Fabaceae</Emphasis>plants

Background  

The phloem of dicotyledonous plants contains specialized P-proteins (phloem proteins) that accumulate during sieve element differentiation and remain parietally associated with the cisternae of the endoplasmic reticulum in mature sieve elements. Wounding causes P-protein filaments to accumulate at the sieve plates and block the translocation of photosynthate. Specialized, spindle-shaped P-proteins known as forisomes that undergo reversible calcium-dependent conformational changes have evolved exclusively in the Fabaceae. Recently, the molecular characterization of three genes encoding forisome components in the model legume Medicago truncatula (MtSEO1, MtSEO2 and MtSEO3; SEO = sieve element occlusion) was reported, but little is known about the molecular characteristics of P-proteins in non-Fabaceae.     

Characterization of five subgroups of the sieve element occlusion gene family in Glycine max reveals genes encoding non-forisome P-proteins,forisomes and forisome tails

P-proteins are structural phloem proteins discussed to be involved in the rapid sealing of injured sieve elements. P-proteins are found in all dicotyledonous and some monocotyledonous plants, but additional crystalloid P-proteins, known as forisomes, have evolved solely in the Fabaceae. Both types are encoded by members of the sieve element occlusion (SEO) gene family, which comprises seven phylogenetic subgroups. The Fabaceae-specific subgroup 1 contains genes encoding forisome subunits in e.g. Medicago truncatula, Vicia faba, Dipteryx panamensis and Canavalia gladiata whereas basal subgroup 5 encodes P-proteins in Nicotiana tabacum (tobacco) and Arabidopsis thaliana. The function of remaining subgroups is still unknown. We chose Glycine max (soybean) as a model to investigate SEO proteins representing different subgroups in one species. We isolated native P-proteins to determine the SEO protein composition and analyzed the expression pattern, localization and structure of the G. max SEO proteins representing five of the subgroups. We found that subgroup 1 GmSEO genes encode forisome subunits, a member of subgroup 5 encodes a non-forisome P-protein and subgroup 2 GmSEO genes encode the components of forisome tails, which are present in a restricted selection of Fabaceaen species. We therefore present the first molecular characterization of a Fabaceae non-forisome P-protein and the first evidence that forisome tails are encoded by a phylogenetically-distinct branch of the SEO gene family.     

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.     

The Ca2+ response of a smart forisome protein is dependent on polymerization

Forisomes are giant self‐assembling mechanoproteins that undergo reversible structural changes in response to Ca²⁺ and various other stimuli. Artificial forisomes assembled from the monomer MtSEO‐F1 can be used as smart biomaterials, but the molecular basis of their functionality is not understood. To determine the role of protein polymerization in forisome activity, we tested the Ca²⁺ association of MtSEO‐F1 dimers (the basic polymerization unit) by circular dichroism spectroscopy and microscale thermophoresis. We found that soluble MtSEO‐F1 dimers neither associate with Ca²⁺ nor undergo structural changes. However, polarization modulation infrared reflection absorption spectroscopy revealed that aggregated MtSEO‐F1 dimers and fully‐assembled forisomes associate with Ca²⁺, allowing the hydration of poorly‐hydrated protein areas. A change in the signal profile of complete forisomes indicated that Ca²⁺ interacts with negatively‐charged regions in the protein complexes that only become available during aggregation. We conclude that aggregation is required to establish the Ca²⁺ response of forisome polymers.     

Anisotropic contraction in forisomes: simple models won't fit

Forisomes are ATP-independent, Ca(2+)-driven contractile protein bodies acting as reversible valves in the phloem of plants of the legume family. Forisome contraction is anisotropic, as shrinkage in length is associated with radial expansion and vice versa. To test the hypothesis that changes in length and width are causally related, we monitored Ca(2+)- and pH-dependent deformations in the exceptionally large forisomes of Canavalia gladiata by high-speed photography, and computed time-courses of derived geometric parameters (including volume and surface area). Soybean forisomes, which in the resting state resemble those of Canavalia geometrically but have less than 2% of the volume, were also studied to identify size effects. Calcium induced sixfold volume increases in forisomes of both species; in soybean, responses were completed in 0.15 s, compared to about 0.5 s required for a rapid response in Canavalia followed by slow swelling for several minutes. This size-dependent behavior supports the idea that forisome contractility might rest on similar mechanisms as those of polyelectrolyte gels, a class of artificial "smart" materials. In both species, time-courses of forisome length and diameter were variable and lacked correlation, arguing against a simple causal relationship between changes in length and width. Moreover, changes in the geometry of soybean forisomes differed qualitatively between Ca(2+)- and pH-responses, suggesting that divalent cations and protons target different sites on the forisome proteins.     

Micromechanical measurements on P-protein aggregates (forisomes) from Vicia faba plants

Forisomes are chemomechanically active P-protein aggregates found in the phloem of legumes. They can convert chemical energy into mechanical work when induced by divalent metal ions or changes in pH, which control the folding state of individual forisome proteins. We investigated the changing geometric parameters of individual forisomes and the strength and dynamics of the forces generated during this process. Three different divalent ions were tested (Ca²⁺, Sr²⁺ and Ba²⁺) and were shown to induce similar changes to the normalized length and diameter. In the concentration range from 0.1 to 4 M, K⁺ and Cl^? ions had no influence on the contraction behaviour of the forisomes induced by 10 mM Ca²⁺. In the absence of dissolved oxygen, these changes were independent of the radius of the metal ion, water uptake and the strength of binding between the selected metal ions and those protein molecules responsible for forisome conformational transformation. In the absence of any load, bound Ca²⁺, Sr²⁺ and Ba²⁺ ions showed apparent and averaged dissociation constants of 14, 62 and 1070 µM, respectively. Various forisomes generated bending on a quartz glass fibre with a diameter of 9 µm. The fibre bending was measured microscopically also by correlation between the digital patterns of a predefined window of observation before and after bending. Similar bending forces of approximately 90 nN were measured for a single forisome sequentially exposed to 10 mM Ca²⁺, Sr²⁺ and Ba²⁺. In the absence of dissolved oxygen, the same conditions resulted in averaged bending forces of (93 ± 40) nN, (58 ± 20) nN, and (91 ± 20) nN after contacting different forisomes with 10 mM Ca²⁺, 10 mM Sr²⁺, and 10 mM Ba²⁺ respectively, demonstrating that the force generated was independent on ion concentrations above a certain threshold value.