Phloem flow strongly influences the systemic spread of silencing in GFP Nicotiana benthamiana plants

The term 'RNA silencing' describes a process that results in the specific degradation of an RNA target. In plants, silenced tissues can initiate the spreading of the process into non-silenced regions by a mobile signal that can be transmitted over long distances. In the present work, we made use of a modified grafting approach to elucidate the driving force behind long-distance transport of the silencing signal. We made reciprocal grafts of two GFP-transgenic Nicotiana benthamiana lines, the non-silenced line 16c (sensor) and the silenced line 6.4 (inducer). We show that the direction of systemic spread of silencing from inducer to sensor can be manipulated by altering sink/source relations in the plant. Using radioactive phosphate as a phloem tracer, we demonstrated that plants that transmitted silencing from silenced scion to non-silenced rootstock had developed a persisting phloem flow from scion to rootstock. These data provide experimental proof of what has been hypothesized so far, that the silencing signal travels via phloem from source to sink. We present here evidence that the appearance of systemic silencing is not an accidental stochastic process, but can be predicted on the basis of the direction of phloem flow.     

Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus. A role for thiol-peptides in the long-distance transport of cadmium and the effect of cadmium on iron translocation

Phytochelatins (PCs) are glutathione-derived peptides that function in heavy metal detoxification in plants and certain fungi. Recent research in Arabidopsis has shown that PCs undergo long-distance transport between roots and shoots. However, it remains unknown which tissues or vascular systems, xylem or phloem, mediate PC translocation and whether PC transport contributes to physiologically relevant long-distance transport of cadmium (Cd) between shoots and roots. To address these questions, xylem and phloem sap were obtained from Brassica napus to quantitatively analyze which thiol species are present in response to Cd exposure. High levels of PCs were identified in the phloem sap within 24 h of Cd exposure using combined mass spectrometry and fluorescence HPLC analyses. Unexpectedly, the concentration of Cd was more than four-fold higher in phloem sap compared to xylem sap. Cadmium exposure dramatically decreased iron levels in xylem and phloem sap whereas other essential heavy metals such as zinc and manganese remained unchanged. Data suggest that Cd inhibits vascular loading of iron but not nicotianamine. The high ratios [PCs]/[Cd] and [glutathione]/[Cd] in the phloem sap suggest that PCs and glutathione (GSH) can function as long-distance carriers of Cd. In contrast, only traces of PCs were detected in xylem sap. Our results suggest that, in addition to directional xylem Cd transport, the phloem is a major vascular system for long-distance source to sink transport of Cd as PC–Cd and glutathione–Cd complexes.     

Long distance transport and movement of RNA through the phloem

Cell-to-cell communication is essential for plant development and adaptation to environmental changes. As a strategy for efficient intercellular communication, plants have evolved a plant-specific symplasmic network connected via plasmodesmata that allows a locally restricted information exchange from cell to cell. A rapid information transfer over long distances is enabled via the phloem transport tubes that pervade the complete plant and thus connect even the most distant organs. While communication by small molecules like metabolites and phytohormones is comparably well studied, the intercellular trafficking of proteins and RNAs has only recently emerged as a novel mechanism of cell-to-cell and long-distance signalling in plants. In particular the non-cell-autonomous and systemic transport pathway for specific RNAs seems to play a key role in the co-ordination of important physiological processes, including virus defence, gene silencing, regulation of development, and nutrient allocation. This review is a summary of the current knowledge on RNAs contained in the phloem long-distance transport system, their transport mechanism, and their potential functions.     

Phloem unloading via the apoplastic pathway is essential for shoot distribution of root-synthesized cytokinins

Root-synthesized cytokinins are transported to the shoot and regulate the growth, development, and stress responses of aerial tissues. Previous studies have demonstrated that Arabidopsis (Arabidopsis thaliana) ATP binding cassette (ABC) transporter G family member 14 (AtABCG14) participates in xylem loading of root-synthesized cytokinins. However, the mechanism by which these root-derived cytokinins are distributed in the shoot remains unclear. Here, we revealed that AtABCG14-mediated phloem unloading through the apoplastic pathway is required for the appropriate shoot distribution of root-synthesized cytokinins in Arabidopsis. Wild-type rootstocks grafted to atabcg14 scions successfully restored trans-zeatin xylem loading. However, only low levels of root-synthesized cytokinins and induced shoot signaling were rescued. Reciprocal grafting and tissue-specific genetic complementation demonstrated that AtABCG14 disruption in the shoot considerably increased the retention of root-synthesized cytokinins in the phloem and substantially impaired their distribution in the leaf apoplast. The translocation of root-synthesized cytokinins from the xylem to the phloem and the subsequent unloading from the phloem is required for the shoot distribution and long-distance shootward transport of root-synthesized cytokinins. This study revealed a mechanism by which the phloem regulates systemic signaling of xylem-mediated transport of root-synthesized cytokinins from the root to the shoot.

Phloem unloading via the apoplastic pathway is essential for shoot distribution and long-distance translocation of root-synthesized cytokinins from the root to the shoot through the xylem.     

Cell-to-Cell, but not long-distance, spread of RNA silencing that is induced in individual epidermal cells

A Turnip crinkle virus (TCV)-based system was devised to discriminate cell-to-cell and systemic long-distance spread of RNA silencing in plants. Modified TCV-GFPDeltaCP, constructed by replacing the coat protein (CP) gene with the green fluorescent protein (GFP) gene, replicated in single epidermal cells but failed to move from cell to cell in Nicotiana benthamiana. Mechanical inoculation of TCV-GFPDeltaCP induced effective RNA silencing in single epidermal cells which spread from cell to cell to form silenced foci on inoculated leaves, but no long-distance systemic spread of RNA silencing occurred. Agroinfiltration of TCV-GFPDeltaCP was, however, able to induce both local and systemic RNA silencing. TCV coinfection arrested TCV-GFPDeltaCP-mediated local induction of RNA silencing. Possible mechanisms involved in cell-to-cell and long-distance spread of RNA silencing are discussed.     

Long-distance movement factor: a transport function of the potyvirus helper component proteinase.

Transport of viruses from cell to cell in plants typically involves one or more viral proteins that supply dedicated movement functions. Transport from leaf to leaf through phloem, or long-distance transport, is a poorly understood process with requirements differing from those of cell-to-cell movement. Through genetic analysis of tobacco etch virus (TEV; potyvirus group), a novel long-distance movement factor was identified that facilitates vascular-associated movement in tobacco. A mutation in the central region of the helper component proteinase (HC-Pro), a TEV-encoded protein with previously described activities in aphid-mediated transmission and polyprotein processing, inactivated long-distance movement. This mutant virus exhibited only minor defects in genome amplification and cell-to-cell movement functions. In situ histochemical analysis revealed that the mutant was capable of infecting mesophyll, bundle sheath, and phloem cells within inoculated leaves, suggesting that the long-distance movement block was associated with entry into or exit from sieve elements. The long-distance movement defect was specifically complemented by HC-Pro supplied in trans by a transgenic host. The data indicate that HC-Pro functions in one or more steps unique to long-distance transport.     

A systemic small RNA signaling system in plants

Systemic translocation of RNA exerts non-cell-autonomous control over plant development and defense. Long-distance delivery of mRNA has been proven, but transport of small interfering RNA and microRNA remains to be demonstrated. Analyses performed on phloem sap collected from a range of plants identified populations of small RNA species. The dynamic nature of this population was reflected in its response to growth conditions and viral infection. The authenticity of these phloem small RNA molecules was confirmed by bioinformatic analysis; potential targets for a set of phloem small RNA species were identified. Heterografting studies, using spontaneously silencing coat protein (CP) plant lines, also established that transgene-derived siRNA move in the long-distance phloem and initiate CP gene silencing in the scion. Biochemical analysis of pumpkin (Cucurbita maxima) phloem sap led to the characterization of C. maxima Phloem SMALL RNA BINDING PROTEIN1 (CmPSRP1), a unique component of the protein machinery probably involved in small RNA trafficking. Equivalently sized small RNA binding proteins were detected in phloem sap from cucumber (Cucumis sativus) and lupin (Lupinus albus). PSRP1 binds selectively to 25-nucleotide single-stranded RNA species. Microinjection studies provided direct evidence that PSRP1 could mediate the cell-to-cell trafficking of 25-nucleotide single-stranded, but not double-stranded, RNA molecules. The potential role played by PSRP1 in long-distance transmission of silencing signals is discussed with respect to the pathways and mechanisms used by plants to exert systemic control over developmental and physiological processes.     

RNA interference-mediated gene knockdown within specific cell types

In plants, RNA interference (RNAi)-induced gene silencing can spread from the initiation site to nearby cells. The silencing signal moves from cell-to-cell through plasmodesmata and, over long distances, through the phloem. In this study, we employed a nuclear-localized GFP fusion protein to visualize the pattern of gene silencing induced by three different transgenes expressing double-stranded RNA (dsRNA) in Arabidopsis root tips. In all cases, we found that dsRNA-induced silencing did not spread from the silencing initiation site to adjacent cells. In the first set of experiments, in a transgenic background expressing nuclear-localized GFP within a contiguous cell layer that included endodermis, cortex/endodermis (joint) initial (CEI) cells and the quiescent center (QC) cells, expression of the marker gene was silenced specifically in the QC cells without affecting gene expression in the adjacent CEI and endodermal cells. The next two sets of experiments examined the knockdown of two endogenous genes. We observed that silencing was completely restricted to the QC and endodermal cells within which the dsRNA transgenes were expressed. Overall, these results accentuate one important aspect of RNAi-induced gene silencing, that it can be cell autonomous, and demonstrated the feasibility of selective gene knockdown within specific cell types.     

RNA silencing movement in plants.

Higher eukaryotes have developed a mechanism of sequence-specific RNA degradation which is known as RNA silencing. In plants and some animals, similar to the nematode Caenorhabditis elegans, RNA silencing is a non-cell-autonomous event. Hence, silencing initiation in one or a few cells leads progressively to the sequence-specific suppression of homologous sequences in neighbouring cells in an RNA-mediated fashion. Spreading of silencing in plants occurs through plasmodesmata and results from a cell-to-cell movement of a short-range silencing signal, most probably 21-nt siRNAs (short interfering RNAs) that are produced by one of the plant Dicer enzymes. In addition, silencing spreads systemically through the phloem system of the plants, which also translocates metabolites from source to sink tissues. Unlike the short-range silencing signal, there is little known about the mediators of systemic silencing. Recent studies have revealed various and sometimes surprising genetic elements of the short-range silencing spread pathway, elucidating several aspects of the processes involved. In this review we attempt to clarify commonalities and differences between the individual silencing pathways of RNA silencing spread in plants.     

Interaction between long-distance transport factor and Hsp70-related movement protein of Beet yellows virus

Systemic spread of viruses in plants involves local movement from cell to cell and long-distance transport through the vascular system. The cell-to-cell movement of the Beet yellows virus (BYV) is mediated by a movement protein that is an Hsp70 homolog (Hsp70h). This protein is required for the assembly of movement-competent virions that incorporate Hsp70h. By using the yeast two-hybrid system, in vitro coimmunoprecipitation, and in planta coexpression approaches, we show here that the Hsp70h interacts with a 20-kDa BYV protein (p20). We further demonstrate that p20 is associated with the virions presumably via binding to Hsp70h. Genetic and immunochemical analyses indicate that p20 is dispensable for assembly and cell-to-cell movement of BYV but is required for the long-distance transport of virus through the phloem. These results reveal a novel activity for the Hsp70h that provides a molecular link between the local and systemic spread of a plant virus by docking a long-distance transport factor to virions.     

RNA silencing movement in plants

Higher eukaryotes have developed a mechanism of sequence-specific RNA degradation which is known as RNA silencing. In plants and some animals, similar to the nematode Caenorhabditis elegans, RNA silencing is a non-cell-autonomous event. Hence, silencing initiation in one or a few cells leads progressively to the sequence-specific suppression of homologous sequences in neighbouring cells in an RNA-mediated fashion. Spreading of silencing in plants occurs through plasmodesmata and results from a cell-to-cell movement of a short-range silencing signal, most probably 21-nt siRNAs (short interfering RNAs) that are produced by one of the plant Dicer enzymes. In addition, silencing spreads systemically through the phloem system of the plants, which also translocates metabolites from source to sink tissues. Unlike the short-range silencing signal, there is little known about the mediators of systemic silencing. Recent studies have revealed various and sometimes surprising genetic elements of the short-range silencing spread pathway, elucidating several aspects of the processes involved. In this review we attempt to clarify commonalities and differences between the individual silencing pathways of RNA silencing spread in plants.     

Symplastic continuity between companion cells and the translocation stream: long-distance transport is controlled by retention and retrieval mechanisms in the phloem

Substantial symplastic continuity appears to exist between companion cells (CCs) and sieve elements of the phloem, which suggests that small solutes within the CC are subject to indiscriminate long-distance transport via the translocation stream. To test this hypothesis, the distributions of exotic and endogenous solutes synthesized in the CCs of minor veins were studied. Octopine, a charged molecule derived from arginine and pyruvate, was efficiently transported through the phloem but was also transferred in substantial amounts to the apoplast, and presumably other non-phloem compartments. The disaccharide galactinol also accumulated in non-phloem compartments, but long-distance transport was limited. Conversely, sucrose, raffinose, and especially stachyose demonstrated reduced accumulation and efficient transport out of the leaf. We conclude that small metabolites in the cytosol of CCs do enter the translocation stream indiscriminately but are also subject to distributive forces, such as nonselective and carrier-mediated membrane transport and symplastic dispersal, that may effectively clear a compound from the phloem or retain it for long-distance transport. A model is proposed in which the transport of oligosaccharides is an adaptive strategy to improve photoassimilate retention, and consequently translocation efficiency, in the phloem.     

Identification of a new glucosinolate-rich cell type in Arabidopsis flower stalk

Distribution of K, Ca, Cl, S, and P in freeze-dried sections of Arabidopsis flower stalk was analyzed by energy dispersive x-ray imaging. Concentrations of these elements in different cell types were quantified by microanalysis of single-cell samples and phloem exudates. Results showed a differential pattern of distribution for all five elements. K concentration was found to be highest in the parenchymatous tissue around vascular bundles. Ca and Cl were present mainly in the central part of the flower stalk. P was largely located in the bundles and in the parenchyma surrounding them. S signal was extraordinary high in groups of cells (S-cells) situated between the phloem of every vascular bundle and the endodermis. Enzymatic hydrolysis by thioglucosidase of cell sap collected from S-cells using a glass microcapillary resulted in the release of glucose, indicating that these cells contain glucosinolates at high (> 100 mM) concentration, which is consistent with the concentration of S (> 200 mM) estimated by x-ray analysis of cell sap samples. Since their position outside of the phloem is ideally suited for protecting the long-distance transport system from feeding insects, the possible roles of these cells as components of a plant defense system are discussed.     

Why do viruses need phloem for systemic invasion of plants?

Plant viruses use sieve elements in phloem as the route of long-distance movement and systemic infection in plants. Plants, in turn, deploy RNA silencing, R-gene mediated defence and other mechanisms to prevent phloem transport of viruses. Cell-to-cell movement of viruses from an initially infected leaf to stem and other parts of the plant could be another possibility for systemic invasion, but it is considered to be too slow. This idea is supported by observations made on viruses that are deficient in phloem loading. The leaf abscission zone forming at the base of the petiole may constitute a barrier that prevents viral cell-to-cell movement. The abscission zone and protective layer are difficult to localize in the petiole until the leaf reaches an advanced stage of senescence. Viruses tagged with the green fluorescent protein are helpful for localization and study of the developing abscission zone.     

Tobacco mosaic virus movement protein enhances the spread of RNA silencing

Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP) may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA.