Nematode infection triggers the de novo formation of unloading phloem that allows macromolecular trafficking of green fluorescent protein into syncytia

Syncytial feeding complexes induced by the cyst nematode Heterodera schachtii represent strong metabolic sinks for photoassimilates. These newly formed structures were described to be symplastically isolated from the surrounding root tissue and their mechanism of carbohydrate import has repeatedly been under investigation. Here, we present analyses of the symplastic connectivity between the root phloem and these syncytia in nematode-infected Arabidopsis (Arabidopsis thaliana) plants expressing the gene of the green fluorescent protein (GFP) or of different GFP fusions under the control of the companion cell (CC)-specific AtSUC2 promoter. In the same plants, phloem differentiation during syncytium formation was monitored using cell-specific antibodies for CCs or sieve elements (SEs). Our results demonstrate that free, CC-derived GFP moved freely from the phloem into the syncytial domain. No or only marginal cell-to-cell passage of GFP was observed into other root cells adjacent to these syncytia. In contrast, membrane-anchored GFP variants as well as soluble GFP fusions with increased molecular masses were restricted to the SE-CC complex. The presented data also show that nematode infection triggers the de novo formation of phloem containing an approximately 3-fold excess of SEs over CCs. This newly formed phloem exhibits typical properties of unloading phloem previously described in other sink tissues. Our results reveal the existence of a symplastic pathway between phloem CCs and nematode-induced syncytia. The plasmodesmata responsible for this symplastic connectivity allow the cell-to-cell movement of macromolecules up to 30 kD and are likely to represent the major or exclusive path for the supply of assimilates from the phloem into the syncytial complex.     

Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific protein trafficking into sieve elements and identifies a novel post-phloem domain in roots

Transgenic Arabidopsis plants were constructed to express a range of GFP-fusion proteins (36-67 kDa) under the companion cell (CC)-specific AtSUC2 promoter. These plants were used to monitor the trafficking of these GFP-fusion proteins from the CCs into the sieve elements (SEs) and their subsequent translocation within and out of the phloem. The results revealed a large size exclusion limit (SEL) (>67 kDa) for the plasmodesmata connecting SEs and CCs in the loading phloem. Membrane-anchored GFP-fusions and a GFP variant targeted to the endoplasmic reticulum (ER) remained inside the CCs and were used as 'zero trafficking' controls. In contrast, free GFP and all soluble GFP-fusions, moved from the CCs into the SEs and were subsequently translocated through the phloem. Phloem unloading and post-phloem transport of these mobile GFP-fusions were studied in root tips, where post-phloem transport occurred only for the free form of GFP. All of the other soluble GFP-fusion variants were unloaded and restricted to a narrow zone of cells immediately adjacent to the mature protophloem. It appears that this domain of cells, which has a peripheral SEL of about 27-36 kDa, allows protein exchange between protophloem SEs and surrounding cells, but restricts general access of large proteins into the root tip. The presented data provide additional information on phloem development in Arabidopsis in relation to the formation of symplasmic domains.     

Differential distribution of proteins expressed in companion cells in the sieve element-companion cell complex of rice plants

Sieve tubes are comprised of sieve elements, enucleated cells that are incapable of RNA and protein synthesis. The proteins in sieve elements are supplied from the neighboring companion cells through plasmodesmata. In rice plants, it was unclear whether or not all proteins produced in companion cells had the same distribution pattern in the sieve element-companion cell complex. In this study, the distribution pattern of four proteins, beta-glucuronidase (GUS), green fluorescent protein (GFP), thioredoxin h (TRXh) and glutathione S-transferase (GST) were analyzed. The foreign proteins GUS and GFP were expressed in transgenic rice plants under the control of the TRXh gene promoter (PTRXh), a companion cell-specific promoter. Analysis of leaf cross-sections of PTRXh-GUS and PTRXh-GFP plants indicated high accumulation of GUS and GFP, respectively, in companion cells rather than in sieve elements. GUS and GFP were also detected in phloem sap collected from leaf sheaths of the transgenic rice plants, suggesting these proteins could enter sieve elements. Relative amounts of GFP and endogenous phloem proteins, TRXh and GST, in phloem sap and total leaf extracts were compared. Compared to TRXh and GST, GFP content was higher in total leaf extracts, but lower in phloem sap, suggesting that GFP accumulated mainly in companion cells rather than in sieve elements. On the other hand, TRXh and GST appeared to accumulate in sieve elements rather than in companion cells. These results indicate the evidence for differential distribution of proteins between sieve elements and companion cells in rice plants.     

Structural and functional vein maturation in developing tobacco leaves in relation to AtSUC2 promoter activity

Transgenic tobacco (Nicotiana tabacum) plants expressing green fluorescent protein (GFP) from the AtSUC2 promoter were used to study the function of different vein classes in developing leaves. In sink leaves, unloading capacity occurred acropetally, with the class I (midrib) and class II veins becoming functional in phloem unloading before the maturation of the class III veinal network. In contrast, in developing cotyledons and source leaves, loading capacity occurred in a basipetal direction. There was a strong correlation between loading capacity, as assessed by (14)C Suc uptake and companion cell expression of AtSUC2-GFP. Developing cotyledons were shown to utilize all available vein classes for loading. A second line of transgenic plants was produced in which GFP, expressed from the AtSUC2 promoter, was targeted to the endoplasmic reticulum instead of the cytoplasm. In these AtSUC2-GFP-ER plants, GFP was unable to traffic into the sieve element and was restricted solely to the companion cells of source leaf tissues. Partial shading of leaves undergoing the sink-source transition demonstrated that the activation of the AtSUC2 promoter in tobacco was influenced by light. Functional and structural maturation of the minor veins required light or a product of light. The activation of the AtSUC2 promoter within major veins appears to be regulated differently from that in the minor veins. The relationship between AtSUC2 activation and the activity of endogenous tobacco Suc transporters is discussed.     

Minor vein structure and sugar transport in Arabidopsis thaliana

Leaf and minor vein structure were studied in Arabidopsis thaliana (L.) Heynh. to gain insight into the mechanism(s) of phloem loading. Vein density (length of veins per unit leaf area) is extremely low. Almost all veins are intimately associated with the mesophyll and are probably involved in loading. In transverse sections of veins there are, on average, two companion cells for each sieve element. Phloem parenchyma cells appear to be specialized for delivery of photoassimilate from the bundle sheath to sieve element-companion cell complexes: they make numerous contacts with the bundle sheath and with companion cells and they have transfer cell wall ingrowths where they are in contact with sieve elements. Plasmodesmatal frequencies are high at interfaces involving phloem parenchyma cells. The plasmodesmata between phloem parenchyma cells and companion cells are structurally distinct in that there are several branches on the phloem parenchyma cell side of the wall and only one branch on the companion cell side. Most of the translocated sugar in A. thaliana is sucrose, but raffinose is also transported. Based on structural evidence, the most likely route of sucrose transport is from bundle sheath to phloem parenchyma cells through plasmodesmata, followed by efflux into the apoplasm across wall ingrowths and carrier-mediated uptake into the sieve element-companion cell complex. Received: 5 October 1999 / Accepted: 20 November 1999     

Wounding enhances expression of AtSUC3, a sucrose transporter from Arabidopsis sieve elements and sink tissues

The Arabidopsis AtSUC3 gene encodes a sucrose (Suc) transporter that differs in size and intron number from all other Arabidopsis Suc transport proteins. Each plant species analyzed so far possesses one transporter of this special type, and several functions have been discussed for these proteins, including the catalysis of transmembrane Suc transport, and also Suc sensing and regulation of other Suc transporters. Here, we show that the AtSUC3 protein is localized in the sieve elements of the Arabidopsis phloem and is not colocalized with the companion cell-specific AtSUC2 phloem loader. Even stronger AtSUC3 expression is observed in numerous sink cells and tissues, such as guard cells, trichomes, germinating pollen, root tips, the developing seed coat, or stipules. Moreover, AtSUC3 expression is strongly induced upon wounding of Arabidopsis tissue. The physiological role of AtSUC3 in these different cells and tissues is discussed.     

Phloem unloading in the potato tuber. Pathways and sites of ATPase

Summary Potential pathways for sucrose unloading in the potato tuber were examined by light and electron microscopy. Abundant plasmodesmata connected sieve elements with surrounding parenchyma elements and also sieve elements with companion cells. Plasmodesmata were rarer, however, between companion cells and parenchyma elements. These observations suggest that sucrose may leave the sieve elements and enter the storage parenchyma cells directly via the symplast and that transport through the companion cell may not be a prerequisite for unloading. Plasmodesmata, grouped together in primary pit fields, were also abundant between storage cells, and isolated storage cells, separated enzymically, showed considerable variation in plasmodesmatal distribution between cells and also on different faces of a single cell. Deposition of starch was found to occur in the tuber cortex while an endodermis with Casparian strip was present external to the phloem, suggesting that assimilates initially enter the cortical storage cells by an entirely symplastic pathway. The possible involvement of ATPase in the unloading process was examined cytochemically, using a lead-salt precipitation method. By contrast with previous findings for phloem no evidence was found for ATPase activity that was unique to the sieve element-companion cell complex. The present observations favour the view that phloem unloading in the potato tuber is a symplastic and passive process.     

Plasmodesma-mediated selective protein traffic between "symplasmically isolated" cells probed by a viral movement protein

Intercellular communication is essential for differentiation and development. In plants, plasmodesmata (PD) form cytoplasmic channels for direct communication. During plant development, programmed reduction in PD number and transport capacity creates the so-called symplasmic domains. Small fluorescent dyes and ions can diffuse among cells within a domain but not across domain boundaries. Such symplasmic isolation is thought to allow groups of cells to differentiate and develop into tissues with distinct structures and functions. Whether or how "symplasmically isolated" cells communicate with one another is poorly understood. One well-documented symplasmic domain is the sieve element-companion cell (SE-CC) complex in the phloem tissue. We report here that, when produced in the CC of transgenic tobacco, the 3a movement protein (3a MP) of Cucumber mosaic virus fused to green fluorescent protein (GFP) can traffic out of the SE-CC complex via PD. The extent of 3a MP:GFP traffic across the boundary between vascular and nonvascular tissues depends on organ type and developmental stage. Our findings provide experimental evidence that endogenous machinery exists for protein traffic between the symplasmically isolated SE-CC complex and neighboring cells. We suggest that PD-mediated traffic of selected macromolecules can be a mechanism for symplasmically isolated cells to communicate with one another.     

Localization of ATPase in Sink Tissues of Ricinus

Histochemical localization of ATPase was carried out on phloemtissues from vegetative and reproductive sinks of Ricinus communis,using lead precipitation procedures. Reaction products werelocalized mainly at the plasma membrane of the sieve elements,companion cells and phloem parenchyma cells. Activity was alsopresent in plasmodesmata, the tonoplast of companion cells anddispersed P-protein within the sieve element lumen. The resultsare discussed in relation to the possible involvement of a plasmamembrane ATPase in apoplastic and symplastic unloading fromthe phloem conducting tissues. ATPase, sink tissues, unloading, Ricinus communis     

黄瓜韧皮部的类血影蛋白

以黄瓜 (CucumissativusL .)叶柄为实验材料 ,应用胶体金免疫电镜技术证明类血影蛋白存在于韧皮部的筛管_伴胞复合体中 ,广泛分布于筛分子中的韧皮蛋白纤丝以及筛分子网络结构上 ,并且分布在伴胞的细胞质和线粒体膜以及筛分子与伴胞之间的分支状胞间连丝上 ,表明该蛋白可能由伴胞合成并经由二者之间的胞间连丝运输到筛分子中。用免疫印迹技术证明 ,黄瓜韧皮部汁液蛋白中存在类血影蛋白 ,其分子量约为 2 6 0kD ,与动物细胞中血影蛋白的分子量接近     

Viral infection enables phloem loading of GFP and long-distance trafficking of the protein

It is generally accepted that viral systemic infection follows the source-to-sink symplastic pathway of sugar translocation. In plants that are classified as apoplastic loaders, the boundary between the companion cell-sieve element (CC-SE) complex and neighboring cells is symplastically restricted, and the potential passage of macromolecules between the two domains has yet to be explored. Transgenic tobacco plants expressing green fluorescence protein (GFP) and cucumber mosaic virus (CMV)-encoded proteins fused to GFP under the control of the fructose-1,6-bisphosphatase (FBPase) promoter were produced in order to localize the encoded proteins in mesophyll and bundle sheath cells and to explore the influence of viral infection on the functioning of plasmodesmata interconnecting the two domains. GFP produced outside the vascular tissue could overcome the symplastic barrier between the CC-SE complex and the surrounding cells to enter the vasculature in CMV-infected plants. Grafting of control (non-transgenic) tobacco scions to CMV-infected FBPase-GFP-expressing root stocks confirmed that GFP could move long distances in the phloem. No movement of the gfp mRNA was noticeable in this set of experiments. The ability of GFP to enter the vasculature and move long distances was also evident upon infection of the grafting plants with other viruses. These results provide experimental evidence for alteration of the functioning of plasmodesmata interconnecting the CC-SE complex and neighboring cells by viral infection to enable non-selective trafficking of macromolecules from the mesophyll into the sieve tube.     

A plasma membrane-anchored fluorescent protein fusion illuminates sieve element plasma membranes in Arabidopsis and tobacco

Rapid acquisition of quantitative anatomical data from the sieve tubes of angiosperm phloem has been confounded by their small size, their distance from organ surfaces, and the time-consuming nature of traditional methods, such as transmission electron microscopy. To improve access to these cells, for which good anatomical data are critical, a monomeric yellow fluorescent protein (mCitrine) was N-terminally fused to a small (approximately 6 kD) membrane protein (AtRCI2A) and stably expressed in Arabidopsis thaliana (Columbia-0 ecotype) and Nicotiana tabacum ('Samsun') under the control of a companion cell-specific promoter (AtSUC2p). The construct, called by its abbreviation SUmCR, yielded stable sieve element (SE) plasma membrane fluorescence labeling, even after plastic (methacrylate) embedding. In conjunction with wide-field fluorescence measurements of sieve pore number and position using aniline blue-stained callose, mCitrine-labeled material was used to calculate rough estimates of sieve tube-specific conductivity for both species. The SUmCR construct also revealed a hitherto unknown expression domain of the AtSUC2 Suc-H(+) symporter in the epidermis of the cell division zone of developing root tips. The success of this construct in targeting plasma membrane-anchored fluorescent proteins to SEs could be attributable to the small size of AtRCI2A or to the presence of other signals innate to AtRCI2A that permit the protein to be trafficked to SEs. The construct provides a hitherto unique entrée into companion cell-to-SE protein targeting, as well as a new tool for studying whole-plant phloem anatomy and architecture.     

Long-distance trafficking of macromolecules in the phloem

The fact that macromolecules such as proteins and mRNAs overcome the symplastic barriers between various tissue domains was first evidenced by the movement of plant viruses. We have recently demonstrated that viral infection disengages the symplastic restriction present between the sieve element-companion cell complex and neighboring cells in tobacco plants. As a result, green fluorescent protein, which was produced in mesophyll and bundle sheath cells, could traffic into the sieve tube and travel long distances within the vascular system. In this addendum we discuss the likely existence of a novel plant communication network in which macromolecules also act as long-distance trafficking signals. Plasmodesmata interconnecting sieve elements and companion cells as well as plasmodesmata connecting the sieve tube with neighboring cells may play a central role in establishing this communication network.Key words: companion cells, cucumber mosaic virus, Cucumis melo, plasmodesmata, movement protein, sieve-elementsTranslocation of photoassimilates from the source (site of synthesis) to various sink organs is governed, in part, by short-distance intercellular transfer of assimilates to the loading region of the phloem and long-distance transport within the plant vascular system. Sucrose, which is synthesized in the leaf mesophyll, moves cell-to-cell symplastically through plasmodesmata until it reaches the boundary of the sieve element (SE)-companion cell (CC) complex. In many plant species, the connection between phloem parenchyma (PP)/bundle sheath (BS) cells and CCs is characterized by a sparseness of plasmodesmata (e.g., Solanaceae), and sucrose is exported out of the cells to the apoplast. This type of plants (apoplastic loaders) uses sucrose proton symporters to load the sucrose into the vasculature.¹ Cucurbits are considered one of the model plants for symplastic phloem loading.² This type of plant is characterized by abundant plasmodesmata interconnecting the intermediary cells, which are specialized CCs, with the neighboring BS cells. It is generally accepted that in these plants, phloem loading includes intercellular movement of sucrose through the plasmodesmata, along the entire pathway from the mesophyll cell to the SE-CC complex.Interestingly, the existence of plasmodesmata interconnecting the SE-CC complex and neighboring cells is evident in all plant species that are characterized by an apoplastic phloem-loading mechanism. Moreover, microinjection experiments have indicated that plasmodesmata interconnecting the PP-CC are functional, in that they allow the exchange of small membrane-impermeable fluorescent probes.³ Virus movement through plasmodesmata from the mesophyll into the SEs further supports the notion that the symplastic communication between the CC-SE complex and the neighboring cells is functional.⁴One can assume that in apoplastic-loading plants, it would be an advantage to maintain the SE-CC complex as an isolated domain, with no functional plasmodesmata interconnecting it to the neighboring tissue. Symplastic continuity between the two domains could result in leakage of sucrose out of the vasculature and a significant reduction in the efficacy of sucrose loading. The fact that the two domains are interconnected suggests that any back-leakage of sucrose that might occur is insignificant relative to the likely efficacy of this communication route.What might the advantage be for symplastic communication between the SE-CC complex and the neighboring tissue? Accumulated evidence suggests that at the tissue/organ level, cell-to-cell trafficking of information molecules allows for noncell-autonomous control over a range of processes, whereas at the organismal level, the phloem serves as an information superhighway, delivering a wide range of macromolecules to enable the plant to function as a whole organism.^5–8 We advanced the hypothesis that plasmodesmata interconnecting the CCs and PP/BS cells play a pivotal role in controlling the long-distance trafficking of putative signaling molecules.     

Nonspecific intercellular protein trafficking probed by green-fluorescent protein in plants

Summary Plasmodesmata mediate intercellular transport of proteins, nucleic acids, and small molecules in plants. We show that transiently produced green-fluorescent protein (GFP) trafficked intercellularly in the epidermis of sink leaves, but not of source leaves, in tobacco and cucumber. In contrast, the protein did not traffic in either sink or source leaves of tomato. On the other hand, the protein spread extensively from cell to cell in the epidermis of all leaves and stems ofArabidopsis thaliana as well as in young hypocotyls and cotyledons of tomato and cucumber. GFP could traffic from epidermis to ground tissues in hypocotyls but not in cotyledons of cucumber. GFP fused to a number of mutant forms of the cucumber mosaic virus 3a movement protein (CMV 3a MP) failed to traffic from cell to cell, suggesting that GFP does not have a specific motif for plasmodesmal trafficking. Our data, together with previous findings, indicate that plasmodesmata can mediate both specific and nonspecific intercellular trafficking of proteins. Furthermore, our data suggest that nonspecific protein trafficking is controlled by species-, development-, organ-, and tissue-specific factors. Since GFP can readily traffic from cell to cell, it raises the questions of how metabolites are compartmentalized intercellularly in a plant and of whether some endogenous plant proteins traffic nonspecifically from cell to cell to perform physiological functions yet to be elucidated.Abbreviations CMV cucumber mosaic virus - GFP green-fluorescent protein - MP movement protein - SEL size exclusion limit     

Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells

Background

Plant phloem consists of an interdependent cell pair, the sieve element / companion cell complex. Sucrose transporters are localized to enucleate sieve elements (SE), despite being transcribed in companion cells (CC). Due to the high turnover of SUT1, sucrose transporter mRNA or protein must traffic from CC to SE via the plasmodesmata. Localization of SUT mRNA at plasmodesmatal orifices connecting CC and SE suggests RNA transport, potentially mediated by RNA binding proteins. In many organisms, polar RNA transport is mediated through RNA binding proteins interacting with the 3'-UTR and controlling localized protein synthesis. To study mechanisms for trafficking of SUT1, GFP-fusions with and without 3'-UTR were expressed in transgenic plants.

Results

In contrast to plants expressing GFP from the strong SUC2 promoter, in RolC-controlled expression GFP is retained in companion cells. The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs. Fusion of GFP to SUT1 did however lead to accumulation of SUT1-GFP in the CC, indicating that trafficking was blocked while translational inhibition of SUT1 mRNA was released in CCs.

Conclusion

A fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC. The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone. It is conceivable that SUT1-GFP protein transport through PD to SE was blocked due to the presence of GFP, resulting in retention in CC particles. Alternatively, SUT1 mRNA transport through the PD could have been blocked due to insertion of GFP between the SUT1 coding sequence and 3'-UTR.

     

A morphometric analysis of the phloem-unloading pathway in developing tobacco leaves

A morphometric analysis of developing leaves of Nicotiana tabacum L. was conducted to determine whether imported photoassimilates could be unloaded by symplastic transport and whether interruption of symplastic transport could account for termination of import. Five classes of veins were recognized, based on numbers of cells in transverse section. Photoassimilate is unloaded primarily from Class III veins in tissue nearing the end of the sink phase of development. Smaller veins (Class IV and V) do not transport or unload photoassimilate in sink tissue because the sieve elements of these veins are immature until after the tissue stops importing. In Class III veins the sieve element-companion cell (SE-CC) complexes are surrounded by phloem parenchyma which abuts the bundle sheath. Along the most obvious unloading route, from SE-CC complex to phloem parenchyma to bundle sheath to mesophyll cells, the frequency of plasmodesmata at each interface increases. To determine whether this pattern of plasmodesmatal contact is consistent with symplastic unloading we first demonstrated, by derivation from Fick's law that the rate of diffusion from a compartment is proportional to a number N which is equal to the ratio of surface area to volume of the compartment multiplied by the frequency of pores (plasmodesmata) which connect it to the next compartment. N was calculated for each compartment within the vein which has the SE-CC complex as its center, and was shown to be statistically the same in all cases except one. These observations are consistent with a symplastic unloading route. As the leaf tissue matures and stops importing, plasmodesmatal frequency along the unloading route decreases and contact area between cells also decreases as intercellular spaces enlarge. As a result, the number of plasmodesmata between the SE-CC complex and the first layer of mesophyll cells declines in nonimporting tissue to 34% of the number found in importing tissue, indicating that loss of symplastic continuity between the phloem and surrounding cells plays a role in termination of photoassimilate unloading.Abbreviation SE-CC sieve element-companion cell     

Macromolecular trafficking between Nicotiana tabacum and the holoparasite Cuscuta reflexa

Transgenic tobacco plants expressing green fluorescent protein (GFP) under the control of the companion cell-specific promoter, AtSUC2, were parasitized by the holoparasite Cuscuta reflexa (dodder). GFP, moving in the translocation stream of the host, was transferred to the Cuscuta phloem via the absorbing hyphae of the parasite. An identical pattern of transfer was observed for the phloem-mobile probe, carboxyfluorescein. Following uptake by the parasite, GFP was translocated and unloaded from the Cuscuta phloem in meristematic sink tissues. Contrary to published data, these observations suggest the presence of a functional symplastic pathway between Cuscuta and its hosts, and demonstrate a considerable capacity for macromolecular exchange between plant species.     

A dual switch in phloem unloading during ovule development in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Developing flowers are important sinks in Arabidopsis thaliana. Their energy demand is covered by assimilates which are synthesized in source leaves and transported via the vasculature. Assimilates are unloaded either symplastically through plasmodesmata or apoplastically by specific transport proteins. Here we studied the pathway of phloem unloading and post-phloem transport in developing gynoecia. Using phloem-mobile fluorescent tracers, we show that phloem unloading into cells of ovule primordia followed a symplastic pathway. Subsequently, the same tracers could not move out of phloem cells into mature ovules anymore. A further change in the mode of phloem unloading occurred after anthesis. In open flowers as well as in outgrowing siliques, the phloem was again unloaded via the symplast. This observed onset of symplastic phloem unloading was accompanied by a change in frequency of MP17-GFP-labeled plasmodesmata. We could also show that the change in cell–cell connectivity was independent of fertilization and increasing sink demand. The presented results indicate that symplastic connectivity is highly regulated and varies not only between different sink tissues but also between different developmental stages.