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ANRV375-PP60-10 ARI 13 November 2008 16:2 V I E W A Review in Advance first posted online on November 21, 2008. (Minor changes may still occur before final publication online and in print.) N I N C E S R E D V A Annu. Rev. Plant Biol. 2009.60. Downloaded from arjournals.annualreviews.org by CAS- Institute of Microbiology on 12/02/08. For personal use only. Phloem Transport: Cellular Pathways and Molecular Trafficking Robert Turgeon1 and Shmuel Wolf 2 1 Department of Plant Biology, Cornell University, Ithaca, New York 14853; email: [email protected] 2 The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture and the Otto Warburg Minerva Center for Agricultural Biotechnology, The Hebrew University of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences, Rehovot 76100, Israel; email: [email protected] Annu. Rev. Plant Biol. 2009. 60:207–21 Key Words The Annual Review of Plant Biology is online at plant.annualreviews.org apoplast, companion cell, phloem loading, plasmodesmata, sieve element, symplast This article’s doi: 10.1146/annurev.arplant.043008.092045 c 2009 by Annual Reviews. Copyright All rights reserved 1543-5008/09/0602-0207$20.00 Abstract The phloem transports nutrients, defensive compounds, and informational signals throughout vascular plants. Sampling the complex components of mobile phloem sap is difficult because of the damage incurred when the pressurized sieve tubes are breached. In this review we discuss sampling methods, the artifacts that can be introduced by different sampling procedures, the intricate pathways by which materials enter and exit the phloem, and the major types of compounds transported. Loading and unloading patterns are largely determined by the conductivity and number of plasmodesmata and the position-dependent function of solute-specific, plasma membrane transport proteins. Recent evidence indicates that mobile proteins and RNA are part of the plant’s longdistance communication signaling system. Evidence also exists for the directed transport and sorting of macromolecules as they pass through plasmodesmata. A future challenge is to dissect the molecular and cellular aspects of long-distance macromolecular trafficking in the signal transduction pathways of the whole plant. 207 ANRV375-PP60-10 ARI 13 November 2008 16:2 Annu. Rev. Plant Biol. 2009.60. Downloaded from arjournals.annualreviews.org by CAS- Institute of Microbiology on 12/02/08. For personal use only. Contents INTRODUCTION . . . . . . . . . . . . . . . . . . STRUCTURES AND PATHWAYS. . . SAMPLING THE PHLOEM . . . . . . . . Stylectomy . . . . . . . . . . . . . . . . . . . . . . . . Bleeding Plants . . . . . . . . . . . . . . . . . . . . EDTA-Facilitated Exudation . . . . . . . Carbon Isotope Labeling . . . . . . . . . . . SUBSTANCES TRANSPORTED IN THE PHLOEM . . . . . . . . . . . . . . . Carbohydrates and Amino Acids . . . . Secondary Compounds . . . . . . . . . . . . . Auxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 208 209 210 210 211 212 212 212 212 213 213 214 INTRODUCTION SE: sieve element CC: companion cell Size-exclusion limit (SEL): maximum size of a molecule that can pass through plasmodesmata passively; often based on molecular mass but more properly on its Stokes radius Plasmodesmata-pore unit: a unique structure that has a large channel on the SE side and several branching channels on the CC side Sieve element reticulum: a specialized form of parietal endoplasmic reticulum in sieve elements that is often anastomosing and stacked 208 The phloem plays an inherently integrative role in nutrition, development, and defense. Identifying mobile materials in the phloem is difficult because of limitations in available sampling techniques. Determining how substances enter and exit the phloem is also difficult because of the intricacies of the pathways involved. In this review we consider intercellular routes into and out of the phloem, sampling methods, and recent information on the major types of compounds transported. Owing to space limitations, readers are directed to recent reviews on the subjects of virus movement (61), systemic signaling in plant defense (53, 71), and small RNA transport (61). STRUCTURES AND PATHWAYS The sieve element (SE) forms a functional complex with its associated companion cell (CC). This complex is connected to surrounding phloem parenchyma cells and other cell types by plasmodesmata, primarily via the CC (Figure 1). The conductivity of plasmodesmata that connect CCs to surrounding cells ranges greatly, depending on the tissue type and its stage of development. In some cases Turgeon · Wolf plasmodesmata are numerous and highly permeable and at the other end of the spectrum they are sparse and seemingly closed to solute (Figure 1). Differences in channel size may be difficult to see by electron microscopy unless the pores are large (99). Permeability can be calculated from knowledge of the size-exclusion limit (SEL) (32), but measurements of SEL are difficult in the small and sequestered cells of the phloem. Plasmodesmatal frequencies are sometimes used alone as a rough guide to symplastic continuity, although these numbers can be misleading without information on the SEL (see below). Plasmodesmata are numerous in the phloem of plants that load photoassimilate from the symplast, but they may also be plentiful in some apoplastic loaders (108, 112). Because plasmodesmatal transport is passive, at least for small molecules, as long as a given molecule is in the cytosolic compartment its movement between cells is apparently limited only by its size, the conductivity of the pores, and possibly charge (28). Conversely, apoplastic transport requires efflux into the cell wall and subsequent retrieval by another cell. Such uptake across the plasma membrane can be passive, as in the puzzling “linear” mechanism of solute uptake (21); transporter driven (96); or endocytic (93). The primary communication pathway between the SE and CC is a modified plasmodesmatal complex known as the plasmodesmatapore unit. Green fluorescent protein (GFP) fusion proteins as large as 67 kDa traffic from CCs to SEs (103), indicating that many cytosolic proteins can pass between the two cell types. Within the mature SE, the sieve element reticulum forms a layer of cisternae that covers almost 100% of the plasma membrane, with frequent, circular openings (fenestrations) approximately 80 nm in diameter (102). The space between the plasma membrane and the sieve element reticulum may sequester and shelter molecules from the rushing current of sap. However, the frequency and size of the fenestrations, at least in SEs of cultured tissue (102), indicates that exchange of unanchored molecules into the lumen of the SE could ANRV375-PP60-10 ARI 13 November 2008 16:2 be rapid. Small, clamp-like structures connect membranes of the organelles and sieve element reticulum to each other and to the SE plasma membrane (27) and apparently act as anchors. Annu. Rev. Plant Biol. 2009.60. Downloaded from arjournals.annualreviews.org by CAS- Institute of Microbiology on 12/02/08. For personal use only. SAMPLING THE PHLOEM Phloem sap is notoriously difficult to sample. SEs are under high hydrostatic pressure and because they are connected by large sieve plate pores, sudden pressure release can affect many cells in a series, rupturing organelles and anchoring molecules, disrupting the sieve element reticulum, altering the configuration of phloem protein, plugging the pores, and initiating wound reactions (27). It is also possible that pressure release pulls substances into SEs from other cell types, especially CCs, leading to contamination of exuding sap. Therefore, demonstrating that a substance is present in phloem −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ sap does not prove that it is mobile in the undamaged translocation stream. Even mobile molecules may not have a function. Given the large SEL of the plasmodesmata-pore units, flux between CCs Sieve elements Phloem parenchyma Companion cells PlasmodesmaPlasmodesma= pore unit 1 Cell wall Source Plasmodesma Sieve element reticulum Endoplasmic reticulum 3 2 Sieve plate Figure 1 Primary pathways into and out of the phloem. This hypothetical scheme is a summary of available information and may not pertain in all species or in all tissues. Arrows indicate the direction and approximate degree of net solute movement. In the source leaves of many species, solutes are loaded apoplastically (1) into the companion cells (CC) from phloem parenchyma (PP) cells via specific transporters (blue circles). Where such apoplastic loading occurs, symplastic transport (2) is minimized by reduction in plasmodesmata (pd) numbers and/or permeability. Symplastic loading (2) may or may not involve synthesis of raffinose or stachyose. Both apoplastic and symplastic loading may occur in a given species, but each occurs into different CCs. Flux from CCs to sieve elements (SE) is primarily, if not exclusively, symplastic through highpermeability plasmodesmata-pore units (ppu). Solutes leaked from SEs are retrieved by CCs or other cell types (3). Along the path (conducting) phloem between sources and sinks, the contents of the SEs and CCs exchange readily, but the net direction of flux is out of the phloem to provide surrounding tissues with nutrients. In path phloem, CC plasmodesmata (4) have very low permeability, so efflux is primarily apoplastic. In sink tissues, solute unloading is commonly symplastic (5), but may be apoplastic (6), depending on the type of sink and its stage of development. Specific transporter Path 4 6 Sink 5 www.annualreviews.org • Trafficking in the Phloem 209 ANRV375-PP60-10 ARI 13 November 2008 Annu. Rev. Plant Biol. 2009.60. Downloaded from arjournals.annualreviews.org by CAS- Institute of Microbiology on 12/02/08. For personal use only. Laser-assisted microdissection: a laser is used in conjunction with conventional compound microscopy to manually or robotically harvest specific tissues or cells 16:2 and SEs appears to be indiscriminate for many compounds. These freely exchanged materials are presumably metabolized in sink tissues. Furthermore, where secondary phloem, derived from the vascular cambium, is present, it may be possible for the degraded contents of autolyzing SEs to enter the sap as sieve pores mature. The uses of parasitic plants and various dissection methods, including laser-assisted microdissection (55), are discussed below in the context of the compounds they are used to identify. The more commonly employed sampling approaches are discussed in this section. To date, no method provides a complete and artifact-free picture of the contents of moving phloem sap. Stylectomy Severed stylets of phloem-feeding insects can be used to measure sieve-tube solute composition, solute concentration, turgor pressure, and membrane potential (33, 38, 56). Aphids are most commonly employed, but scales, mealybugs (33), and planthoppers (36) may also be used. Although this technique has been instrumental in phloem research, it has limitations. Stylectomy is technically challenging and restricted to certain plant-insect combinations. Also, exudate quantity is low, and many plants rapidly seal the phloem. It must also be kept in mind that insect-host interactions are biological, as well as mechanical. The very fact that sap exudes suggests that the insect has counteracted sealing defenses. Kennedy & Mittler (63) cautioned that stylet exudate might differ somewhat from sieve-tube sap in an aphid-free plant. Since then researchers have learned that aphids exude saliva, apparently to inhibit plugging (120), and thus they potentially alter sap chemistry (56). Aphid feeding also induces callose deposition (12) and localized changes in gene expression (79). Aphids may also alter sap composition to enhance their diet (106). Although the effects listed above are gross changes resulting from heavy infestations, one wonders if individual aphids induce more sub210 Turgeon · Wolf tle, local changes in the chemical composition of sap. In this regard note that the amino acid compositions of the sap from stylets of different aphid species on the same host plant have been reported to differ (94). Bleeding Plants Some plants, especially trees (39, 127), produce exudate from severed phloem. The cucurbits (23), Ricinus communis (76), and legumes (101) exude copiously. Yucca and palms can provide hundreds of liters of exudate (113). Sap authenticity is often inferred from its expected molecular profile: high concentration of transport carbohydrate, high molar ratio of sugars to amino acids, and little, if any, monosaccharide. However, this reasoning can be circular because it presupposes the results the experiment is designed to obtain. Furthermore, certain phloem contents may not differ much from those of other cell types (Reference 121 and references therein), limiting their usefulness as markers. In practice, the absence of monosaccharides is usually considered the best indicator of phloem sap purity. However, monosaccharides are largely confined to vacuoles, at least in mesophyll cells (80), which means that contamination could originate from the cytosol of nonphloem cells and not include hexose. Physiological correlates can also help identify the exudate source. Sap exudes most profusely from the source side of wounds in Eucalyptus (84), and a source-to-sink gradient also exists in the sugar content of bleeding sap. Some authors have noted diel variation in the sugar concentration of sap (e.g., 77). However, source-to-sink and diel patterns of carbohydrate storage in surrounding, nonphloem cells could also exist. Isotopic labeling can help prove that exudate contains translocated materials (e.g., 100), but this type of analysis does not prove that the exudate is free of unlabeled contaminants. Cucurbits are often used in exudate studies. However, there is reason to question the origin and nature of cucurbit exudate. First, the sugar concentration is sometimes less than Annu. Rev. Plant Biol. 2009.60. Downloaded from arjournals.annualreviews.org by CAS- Institute of Microbiology on 12/02/08. For personal use only. ANRV375-PP60-10 ARI 13 November 2008 16:2 20 mM (31, 89), though in some cases it is closer to 100 mM (77). Cucurbit sap has been suggested to be naturally dilute, but from histochemical analysis (87), plasmolysis studies (110), observations of surging (24), and measurement of sugars in microdissected minor veins (48) it is clear that this is not so. Such studies are consistent with aphid stylectomy measurements of 839 mM sugar in the sap of Alonsoa (Scrophulariaceae) (115), which appears to use the same phloem loading mechanism as cucurbits (109, 115). It could be argued that cucurbit exudate is highly concentrated as it bleeds but is immediately diluted by water from the xylem. This is unlikely given that the water column is under tension (101). Furthermore, dilution does not account for the unexpectedly low concentration of stachyose compared with monosaccharides and amino acids in such sap (31, 89), in direct conflict with radiolabeling experiments (97, 117). Perhaps the low osmotic potential and unusual composition of bleeding sap in cucurbits is associated with the presence of an extrafascicular system of anastomosing phloem strands around the periphery of the bundles and in the cortex (23, 24, 30). The sieve pores of extrafascicular, but not fascicular, SEs are open in severed tissue (24), perhaps explaining why exudate is so copious. Eschrich and coworkers (29) reported that exuding sap originates from vascular bundles and from extrafascicular phloem in the cortex. Therefore, cucurbit sap may be a mixture of the contents of the fascicular and extrafascicular systems. If the extrafascicular system is not part of the long-distance transport network, some of the material in cucurbit exudate could be stationary in unsevered tissue. Unfortunately, little is known about the role of extrafascicular phloem in cucurbits. Radiolabel is present in extrafascicular phloem following 14 CO2 fixation in the lamina (117), suggesting that it is part of the long-distance transport system, but this radiolabel may simply represent sugars exchanging between the two systems. Another possible role for extrafascicular phloem is that of a biosynthetic reservoir (17). EDTA-Facilitated Exudation King & Zeevaart (65) demonstrated that EDTA facilitates exudation of 14 C from cut petioles or shoots after 14 CO2 fixation in the lamina. Their labeling procedure assured that mobile photoassimilates were being sampled. However, the EDTA technique is also used to identify unlabeled compounds, and in this case there is a danger of sampling nonmobile materials. This issue is the same one discussed above for bleeding sap, but it is potentially more serious because EDTA softens the tissue and induces leakage of ions and metabolites, including carbohydrates (54). Darkening leaves during exudation (65) prevents movement of the EDTA into the lamina in the transpiration stream, where it can cause more extensive damage, but the effects on the submerged portion of the petiole cannot be avoided. At higher EDTA concentrations, the proportion of exuded hexose is elevated, consistent with additional leakage (45). There is also a risk that exuded compounds may degrade during the collection period. Hexoses in EDTA exudate are sometimes ascribed to invertase activity. This may or may not be the case, but should not simply be assumed. Problematically, in some studies hexoses are included in the calculation of exuded sucrose, assuming that they arose by hydrolysis. This is a suspect procedure considering that hexoses may have leaked instead from the vacuolar compartment of compromised, nonphloem cells. Discrepancies in metabolite profiles of sap from EDTA-facilitated exudation and aphid stylets have been noted. Fructans and loliose are present in EDTA exudate, but not aphid stylet sap, of Lolium perenne (1), and gamma amino butyric acid is present in EDTA exudate, but not aphid sap, of Medicago sativa (19). In both studies, monosaccharide levels were much higher in the EDTA exudate. Similarly, metabolite studies on Lucerne (41) and oat and barley (118) yielded significant differences when the two methods were compared. When using the EDTA-facilitated exudation technique, identification of novel or unexpected compounds in sap should be corroborated by other methods. www.annualreviews.org • Trafficking in the Phloem 211 ANRV375-PP60-10 ARI 13 November 2008 16:2 The method also does not lend itself to measuring transport rates, because the source/sink balance is drastically altered and the amount exuded is generally much less than that transported in intact plants (65). Carbon Isotope Labeling Annu. Rev. Plant Biol. 2009.60. Downloaded from arjournals.annualreviews.org by CAS- Institute of Microbiology on 12/02/08. For personal use only. In a common and useful isotope labeling approach, leaves are exposed to 14 CO2 and isotopically labeled substances are detected downstream. 11 C can also be employed, but its short half-life precludes its use in most analytical procedures. 14 CO2 labeling was instrumental in early studies that identified transported photoassimilates (9). Labeled compounds can be metabolized in transit, leading to the possible misidentification of metabolic products as transport compounds, but over short periods this effect is minimal (117). Isotopically labeled compounds, such as hormones, can be applied to one region of the plant and detected downstream (e.g., 13). This approach allows researchers to determine if a compound of interest is phloem-mobile, but it does not guarantee that the same endogenous compound is mobile because it may not be in a compartment that makes it available for transport. SUBSTANCES TRANSPORTED IN THE PHLOEM Carbohydrates and Amino Acids All plants transport sucrose, whereas some also transport raffinose and stachyose and/or sugar alcohols, sometimes in greater quantity than sucrose (70, 108). These compounds enter the phloem primarily in mature leaves but also exchange with surrounding tissues along the transport route (5). Numerous plasmodesmata are present in the minor veins of mature leaves in some species (37), providing a potential symplastic pathway for sucrose to enter the phloem passively, down its concentration gradient, as long as there is enough sucrose in the mesophyll to obviate the 212 Turgeon · Wolf need for a concentrating step in the phloem (111). In some other species, such as the cucurbits, sucrose diffuses into the minor vein CCs and is converted there to raffinose and stachyose (74, 108), a polymer trapping mechanism that concentrates the sugars. A third loading mechanism, which is apparently the most common, is through the apoplast. Sucrose effluxes into the apoplast and is then actively loaded into the minor vein phloem by cotransport with protons (70). Some plants apparently load by more than one mechanism, using different CC types in the same veins (108). Less is known about sugar alcohol loading. Sorbitol and mannitol transporters have been localized in the minor vein phloem (Reference 60 and references therein). However, because transporters are also used for retrieval, their presence in the phloem does not prove that loading is apoplastic (108). Sugar alcohol concentrations in the cytosol of Plantago and celery mesophyll cells are much lower than those in aphid stylet exudate (80), providing strong evidence for active, apoplastic loading. However, similar measurements in Prunus produced equivocal results (80). Organic N may enter the phloem by several routes (88). Inorganic N may be carried into mature leaves in the xylem, converted to organic form, and subsequently exported. Organic N may also enter leaves in the xylem to be metabolized, used in protein synthesis, or transferred to the phloem. Comparisons of amino acid concentrations in the mesophyll and phloem (122) and the effects of downregulating an amino acid transporter (66) suggest an active loading mechanism in plants that load sucrose from the apoplast (83). In symplastic loading species, active apoplastic loading may not be required or even possible, because amino acids are smaller than sucrose and should have free access to the phloem via plasmodesmata. Secondary Compounds Many secondary compounds are toxic and are excluded from symplastic compartments, including the phloem. However, the phloem Annu. Rev. Plant Biol. 2009.60. Downloaded from arjournals.annualreviews.org by CAS- Institute of Microbiology on 12/02/08. For personal use only. ANRV375-PP60-10 ARI 13 November 2008 16:2 does transport several types of secondary compounds. Glucosinolates are sulfur-containing glucosides activated by myrosinase (46). Glucosinolates and myrosinase are present in different cellular or intracellular compartments and are mixed by herbivory, releasing toxic products (46). In Arabidopsis, glucosinolate is found in S cells, external to the vascular bundles. Glucosinolates are present in aphid stylet exudate (68), but it is still not known where or how they enter the phloem. Glucosinolate transporters have been characterized (15), and glucosinolate enters the phloem when applied to leaves (16), but transporter localization does not definitively prove that loading of endogenous material is apoplastic (see above). Iridoid glycosides are terpene-derived compounds that protect against nonadapted insect herbivores, fungi, and bacteria (14). Antirrhinoside (44, 115) and catalpol (112) are translocated in Asarina and Catalpa, respectively. Voitsekhovskaja and coworkers (115) measured high concentrations of antirrhinoside (786 mM) in aphid stylet exudate from Asarina. One wonders if such high levels in exudate are due to release of antirrhinoside into the phloem in response to aphid attack. Pyrrolizidine alkaloids (PAs) are defensive compounds (49) and are found in the phloem (123). In Senicio, synthesis of the backbone structure of PA is restricted to roots (78). How PAs are loaded into the phloem and transported from roots to shoots against the typical direction of flow is unknown (78). Complex compartmentation and the translocation of intermediates between cell types are common in alkaloid synthesis (126). Many of these pathways operate close to, if not within, the phloem, perhaps taking advantage of the carbohydrate supply. In opium poppy, alkaloids synthesis is associated with the phloem and the compounds accumulate in laticifers. Whether this synthesis occurs in phloem parenchyma cells and laticifers themselves (119) or in SEs (10) is a matter of contention. Cardenolides are defensive steroid compounds. On the basis of aphid feeding patterns, Botha and colleagues (11) suggested that cardenolides are transported in the internal phloem. These compounds are found in aphid honeydew (20) and Cuscuta growing on Digitalis (91). Auxin Indole-3-acetic acid (IAA) is transported cellto-cell (35) and also in the phloem. Exogenous radioactive IAA migrates at 3–5 cm h−1 (18), 5–10 times faster than cell-to-cell transport (35). Also, endogenous auxin (Aux) is present in phloem sap of Ricinus communis (6) and in sap collected from Pisum sativum leaves using the EDTA-facilitated exudation protocol (59). Interestingly, inhibition of polar Aux transport from shoot apex to roots does not reduce the Aux effect on lateral root development, suggesting that phloem-based transport of IAA is also involved in controlling root architecture (18). Indole-3-acetic acid (IAA): a native form of the plant hormone auxin; involved in regulation of spatial and temporal aspects of plant growth and development Proteins Proteins are present in phloem sap (Reference 107 and references therein). Several hundred proteins have been identified in the sap of cucurbits, Ricinus, and oilseed rape (Reference 98 and references therein). Protein concentration in Ricinus sap is 0.1 to 2 mg ml−1 (47) and up to 100 mg ml−1 in cucurbit exudate (89). Because mature SEs cannot synthesize protein, the most likely origin of phloem-sap proteins is the immature SE or the CC. Considering that GFP-fusion proteins greater than 67 kDa traffic from CCs to SEs in transgenic plants (103), it is reasonable to propose that many proteins in sieve tube sap have nonspecifically diffused (leaked) out of the CCs and into the SEs. Conversely, some proteins in phloem sap are larger than 100 kDa in size (7, 34, 98, 116), and protein profiles of phloem sap are significantly different from those of surrounding cells (e.g., 40, 116). These findings suggest facilitated and regulated protein movement into SEs. Indeed, proteins derived from pumpkin sap have the capacity to alter the SEL of plasmodesmata that interconnect mesophyll cells (7), though www.annualreviews.org • Trafficking in the Phloem 213 ARI 13 November 2008 16:2 evidence on plasmodesmata-pore unit regulation is lacking. Additional evidence suggests that protein trafficking into and out of the SE is regulated. Although the P-proteins of cucurbits, PP1 and PP2, traffic in both directions between CCs and SEs (42, 69), serpin, a defense-related protein smaller than PP1 (43 kDa versus 88 kDa), appears unable to move from SE to CC (69). Protein profiles in SE sap are site specific. For example, new soluble proteins in wheat phloem sap are added and other proteins are removed along the transport pathway (34), more proteins are present in sap collected from large than from small pumpkin fruits (62), and differences are seen in comparisons of proteins in sap collected from different organs of melon plants (D. Malter & S. Wolf, unpublished data). In addition, allene oxide cyclase, a key enzyme in the jasmonate pathway, but not its mRNA, is present in SE plastids in tomato,which suggests synthesis in the CC and directed export to SE plastids (50). It is not known how protein trafficking into and out of SEs is regulated, but viral protein movement may provide clues. For example, virus infection enables the movement of GFP from the mesophyll to the sieve tube (85), and the 3a movement protein of Cucumber mosaic virus fused to GFP can traffic out of the SE-CC complex in tissue where there is no movement of solute through the same plasmodesmata (57). Perhaps viruses exploit an endogenous mechanism for protein trafficking across apparently closed symplastic boundaries. Control over the import and export of proteins from the SEs presumably involves protein modifications inside the sieve tube. The finding that numerous pumpkin phloem-sap proteins can be phosphorylated and/or glycosylated suggests that these proteins are subjected to posttranslational modifications required for their interaction with a chaperone (protein-protein interaction) to enable their trafficking out of the sieve tube (105). A unique motif that facilitates trafficking through plasmodesmata was characterized in a chaperone isolated from Cucurbita maxima phloem sap (2). Annu. Rev. Plant Biol. 2009.60. Downloaded from arjournals.annualreviews.org by CAS- Institute of Microbiology on 12/02/08. For personal use only. ANRV375-PP60-10 214 Turgeon · Wolf Perhaps the most pronounced alteration in plant development is the transition from vegetative to floral growth, which is governed by environmental and internal signals. Longdistance trafficking of the protein product of FLOWERING LOCUS T (FT) is associated with the promotion of flowering in Arabidopsis, pumpkin, and rice (22, 72, 104), which provides strong evidence that FT is the long-sought florigen. The current challenge is to inhibit specific phloem trafficking of FT and explore the influence of this inhibition on flowering. It is logical to assume that long-distance protein trafficking is directed by mass flow from source to sink organs. However, rootward movement of C. maxima PP16-1 apparently depends on its interaction with specific phloem proteins (2, 3). Note, however, that these pioneering studies were based on microinsertion of biotinylated pumpkin protein into a rice sieve tube via cut stylets. Detection of the tracer proteins in the various plant organs might have been due to this ultrasensitive experimental system. RNA Almost 40 years ago, traces of RNA were found in phloem sap, but were considered to be contaminants from neighboring cells (67, 125). More recently, transcripts encoding proteins related to metal homeostasis, stress response, protein degradation or turnover, and phloem structure and metabolism have been identified and characterized in phloem-enriched tissues (86, 114). The laser-assisted microdissection technique has been used to collect clusters of phloem cells that include several cell types for a cDNA library (4) and for microarray experiments (81). However, the technique has not yet yielded samples from single cell types. RNA analysis has been conducted on sap from aphid stylectomy (25, 95), but these studies have provided only limited genomic information. Therefore, sap collection from bleeding plants is still the primary option for high-throughput analysis of phloem-sap transcripts (26, 82, 92). Functional classification Annu. Rev. Plant Biol. 2009.60. Downloaded from arjournals.annualreviews.org by CAS- Institute of Microbiology on 12/02/08. For personal use only. ANRV375-PP60-10 ARI 13 November 2008 16:2 (based on BLAST descriptions) of almost 1000 transcripts identified in phloem sap from cut melon stems showed that the transcripts fall into the following categories: cellular response to hormones and stress (40%); signal transduction, transcriptional control and nucleic acid binding (15%); and defense-associated responses (10%). Interestingly, only 2% of transcripts are related to metabolism (82). In an alternative approach, a CC-specific library was constructed from Arabidopsis cells selected on the basis of GFP expression driven by the AtSUC2 promoter (58). Partial sequencing of 2000 clones revealed that 29% of the genes are associated with redox regulation and 16% with metabolism. The pronounced differences between the profiles of transcripts identified in phloem sap (82) and CCs (58), albeit from different species, suggests that sap RNA is not simply contamination from CCs. Moreover, the inability to detect CC-specific gfp RNA in wild-type scions on transgenic stocks (51, 85) suggests that entry of endogenous mRNA molecules into the sieve tube is sequence specific. Is it true that the macromolecules present in the sieve tube, including mRNA molecules, are in the process of trafficking long distances? Support for this notion has been provided mainly by experiments in which species-specific mRNA molecules pass through unions in heterografts (Reference 82 and references therein) or from host to parasite (90). The latter discovery raises the fascinating possibility of interspecies communication. Not all phloem sap mRNA molecules traffic systemically: Only 6 of 43 randomly selected melon phloem sap transcripts were identified in scions of heterografts (82). Interestingly, three of the six transcripts encoded Aux/IAA and small auxin up RNA (SAUR) proteins. To date it is unclear how many transcripts are phloem mobile, how stationary mRNA is anchored, and most importantly, what biological roles most transcripts play in enucleated SEs or their target sink tissues. Although it has been suggested that mRNA molecules can operate as long-distance signals (73), evidence to date is limited. Transmission of a phenotype across a graft union in conjunction with mRNA movement was first demonstrated with the mouse ears (Me) mutation, where wild-type scions developed octapinnate compound leaves characteristic of Me mutant plants (64). These results were corroborated in one study (52), but only in low-light growth conditions, and were not corroborated in a second study under any light conditions (75). Using another system, Haywood and coworkers (52) demonstrated that transport of gibberellic acid-insensitive ( gai) transcripts in heterografts altered leaflet morphology in wild-type scions, consistent with impaired GA signaling (52). In a third system, trafficking of BEL1-like transcription factor mRNA in grafted plants resulted in enhanced potato tuber formation (8). The results of these studies are highly intriguing. Given the importance of the concept of long-distance RNA signaling and the possibility of sequence-specific RNA sorting (52), these experiments need to be corroborated and extended. In particular, the relevance of systemic RNA signaling to natural environmental responses needs to be established. Although phloem sap contains no detectable RNAse activity (25, 95), the current perception, based on virus movement studies and the identification of RNA binding proteins in phloem sap (43, 124), is that mRNA molecules traffic long distances as ribonucleoprotein (RNP) complexes and not as naked molecules. Because transcripts cannot be translated in the SEs, such movement must require a chaperoning mechanism to load them into the SEs and unload them in their target sinks. The generality of the concept that mRNA molecules act as signals to coordinate developmental processes at the whole-plant level is still under debate. More information is needed on the sites of synthesis of these molecules and the sequence motifs (or chaperoning mechanisms) required for transcripts to enter, exit, and traffic long distances in sieve tubes. Other important questions relate to the synthesis and possible movement and function of the respective proteins in target tissue. A special challenge will www.annualreviews.org • Trafficking in the Phloem Heterograft: a vegetative tissue (upper stem and shoot apex) of a plant from one species that is transplanted on a tissue (lower stem and roots) of a plant from another species Ribonucleoprotein (RNP) complex: a complex that is formed between RNA and protein 215 ANRV375-PP60-10 ARI 13 November 2008 16:2 be to develop experimental systems in which phenotypic changes can be evaluated following specific inhibition of the long-distance movement of each transcript. SUMMARY POINTS 1. Pathways into and out of the phloem are determined primarily by the distribution and permeability of plasmodesmata and the location, activity, and specificity of transmembrane transporters. 2. Because of its elevated hydrostatic pressure, the phloem is difficult to sample without displacing its contents and possibly mobilizing normally anchored materials and pulling substances into the sieve elements (SEs) from surrounding cells. Annu. Rev. Plant Biol. 2009.60. Downloaded from arjournals.annualreviews.org by CAS- Institute of Microbiology on 12/02/08. For personal use only. 3. Several methods have been developed to sample the phloem but all, including insect stylectomy, can introduce artifacts. 4. Carbohydrates are the primary osmotic agents in phloem sap. The mechanisms of sugar and sugar alcohol loading in leaves are species specific and still under investigation. 5. The functions of most proteins in phloem sap are unknown, as are the mechanisms that confer specificity to their intercellular distribution and mobility. 6. Functions have been attributed to a few RNA molecules in phloem sap, but this area of research is in its infancy. DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review. 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