Analysis of protein transport in the Brassica oleracea vasculature reveals protein-specific destinations

We investigated the vascular transport properties of exogenously applied proteins to Brassica oleracea plants and compared their delivery to various aerial parts of the plant with carboxy fluorescein (CF) dye. We identified unique properties for each protein. Alexafluor-tagged bovine serum albumin (Alexa-BSA) and Alexafluor-tagged Histone H1 (Alexa-Histone) moved slower than CF dye throughout the plant. Interestingly, Alexa-Histone was retained in the phloem and phloem parenchyma while Alexa-BSA moved into the apoplast. One possibility is that Alexa-Histone sufficiently resembles plant endogenous proteins and is retained in the vascular stream, while Alexa-BSA is exported from the cell as a foreign protein. Both proteins diffuse from the leaf veins into the leaf lamina. Alexa-BSA accumulated in the leaf epidermis while Alexa-Histone accumulated mainly in the mesophyll layers. Fluorescein-tagged hepatitis C virus core protein (fluorescein-HCV) was also delivered to B. oleracea plants and is larger than Alexa-BSA. This protein moves more rapidly than BSA through the plant and was restricted to the leaf veins. Fluorescein-HCV failed to unload to the leaf lamina. These combined data suggest that there is not a single default pathway for the vascular transfer of exogenous proteins in B. oleracea plants. Specific protein properties appear to determine their destination and transport properties within the phloem.     

Leaf structure and translocation in sugar beet

Anatomical and ultrastructural details of a translocating 10-cm leaf of sugar beet (Beta vulgaris L. var. Klein Wanzleben) were correlated with translocation rate data. The minor veins were found to be 13 times as extensive as the major veins and measure 70 cm/cm² leaf lamina. Measurements disclosed that a 33-μ length of minor vein services 29 mesophyll cells with the result that translocate moves an average of 73 μ or 2.2 cell diameters during transport from mesophyll cells to a minor vein. High-resolution, freeze-dry autoradiography revealed that assimilates accumulate in organelle-rich cells of the minor vein phloem. Correlation of phloem volume and loading rate for minor veins yielded an uptake rate of 735 μmoles of sucrose per g fresh weight of phloem. The arrangement and structural features of minor veins appeared to be consistent with the concept that vein loading precedes translocation.     

Leaf mechanics and herbivory defence: How tough tissue along the leaf body deters growing insect herbivores

Research on herbivory defence often focuses on leaf chemistry but less on how plant mechanical properties like leaf veins deter herbivores. Herbivores often eat tough, complex plant tissue, yet how mechanical properties affect feeding performance as the consumer grows is unclear. We measured the toughness and strength of five types of leaf tissue – the midrib, the secondary and marginal veins and the lamina inside (inner) and outside (outer) the marginal vein – in mature Eucalyptus viminalis and Eucalyptus ovata leaves with punch tests. Leaf veins were, on average, 6.2 times tougher than lamina. Marginal veins were uniformly strong and tough along the leaf body, while midribs were less strong and secondary veins less tough toward leaf tips. We correlated the force required to puncture leaf tissue with the feeding performance of a chewing insect herbivore (the spiny leaf insect, Extatosoma tiaratum (Phasmida)) across four instar stages to explore the role of tough leaf veins as potential feeding barriers. Larvae more often ate less tough leaf tips and tougher tissue as they grew. However, younger larvae were capable of penetrating the tough marginal vein when starved. We suggest tough leaf veins and consumer position along the leaf body influence insect herbivore feeding performance over their lifetime.     

Oxygen isotope enrichment of organic matter in Ricinus communis during the diel course and as affected by assimilate transport

The oxygen isotope composition in leaf water and organic compounds in different plant tissues is useful for assessing the physiological performance of plants in their environment, but more information is needed on Delta(18)O variation during a diel course. Here, we assessed Delta(18)O of the organic matter in leaves, phloem and xylem in stem segments, and fine roots of Ricinus communis during a full diel cycle. Enrichment of newly assimilated organic matter in equilibrium with leaf water was calculated by applying a nonsteady-state evaporative enrichment model. During the light period, Delta(18)O of the water soluble organic matter pool in leaves and phloem could be explained by a 27 per thousand enrichment compared with leaf water enrichment. Leaf water enrichment influenced Delta(18)O of phloem organic matter during the night via daytime starch synthesis and night-time starch remobilization. Phloem transport did not affect Delta(18)O of phloem organic matter. Diel variation in Delta(18)O in organic matter pools can be modeled, and oxygen isotopic information is not biased during transport through the plant. These findings will aid field studies that characterize environmental influences on plant water balance using Delta(18)O in phloem organic matter or tree rings.     

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.     

The pathways of water movement in leaves modified into tents by bats

A number of species of bats modify leaves into tents, which they use as roost-sites. Through this process, some areas of the leaf lamina are damaged or become detached from the midrib. Such injuries do not cause death of the leaf or the detached areas, indicating that water supply to these areas must be maintained. We examined the anatomy of the vascular systems and water transport in the leaves of three species of plants: Heliconia pogonantha L., Manicaria plukenetii Griseb. & H. Wendl., and Cryosophila warcsewiczii (H. Wend.) Bartlett. In altered leaves of all three species, detached areas of the laminae were supplied with water by minor transverse veins branching from the first major parallel vein that remained intact next to the cut. These transverse veins conducted water through single xylem elements of narrow diameter (approximately 10 urn) previously thought to supply water only to mesophyll cells in their immediate vicinity. The short lengths of these veins compensates their high resistance to water flow (a consequence of their small diameter xylem elements), indicating that small transverse veins have a large capacity for water transport. Water typically flowed through transverse veins into detached major and minor parallel veins, filled these parallel veins in both directions (i.e. toward the midrib and the leaf edge), and continued on into subsequent transverse and parallel veins, thereby supplying water to the entire leaf. Water conduction through these small transverse veins could support large areas of leaf lamina, keeping the leaf-tent alive for at least several months. The maintenance of the flow of water and nutrients to areas of leaves detached by bats during the tent-making process increases the longevity and decreases the conspicuousness of leaf-tents, and is likely a key factor in the success of this roosting strategy.     

A long-distance translocatable phloem protein from cucumber forms a ribonucleoprotein complex in vivo with Hop stunt viroid RNA

Viroids are highly structured plant pathogenic RNAs that do not code for any protein, and thus, their long-distance movement within the plant must be mediated by direct interaction with cellular factors, the nature of which is presently unknown. In addition to this type of RNAs, recent evidence indicates that endogenous RNAs move through the phloem acting as macromolecular signals involved in plant defense and development. The form in which these RNA molecules are transported to distal parts of the plant is unclear. Viroids can be a good model system to try to identify translocatable proteins that could assist the vascular movement of RNA molecules. Here, we demonstrate by use of immunoprecipitation experiments, that the phloem protein 2 from cucumber (CsPP2) is able to interact in vivo with a viroid RNA. Intergeneric graft assays revealed that both the CsPP2 and the Hop stunt viroid RNA were translocated to the scion. The translocated viroid is symptomatic in the nonhost scion, indicating that the translocated RNA is functional. The CsPP2 gene was cloned and sequenced. The analysis of its primary structure revealed the existence of a potential double-spaced-RNA-binding motif, previously identified in a set of proteins that bind to highly structured RNAs, which could explain its RNA-binding properties. The possible involvement of this phloem protein in assisting the long-distance movement of the viroid RNA within the plant is discussed.     

<Superscript>11</Superscript>C-imaging: methyl jasmonate moves in both phloem and xylem,promotes transport of jasmonate,and of photoassimilate even after proton transport is decoupled

The long-distance transport and actions of the phytohormone methyl jasmonate (MeJA) were investigated by using the short-lived positron-emitting isotope ¹¹C to label both MeJA and photoassimilate, and compare their transport properties in the same tobacco plants (Nicotiana tabacum L.). There was strong evidence that MeJA moves in both phloem and xylem pathways, because MeJA was exported from the labeled region of a mature leaf in the direction of phloem flow, but it also moved into other parts of the same leaf and other mature leaves against the direction of phloem flow. This suggests that MeJA enters the phloem and moves in sieve tube sap along with photoassimilate, but that vigorous exchange between phloem and xylem allows movement in xylem to regions which are sources of photoassimilate. This exchange may be enhanced by the volatility of MeJA, which moved readily between non-orthostichous vascular pathways, unlike reports for jasmonic acid (which is not volatile). The phloem loading of MeJA was found to be inhibited by parachloromercuribenzenesulfonic acid (PCMBS) (a thiol reagent known to inhibit membrane transporters), and by protonophores carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP) suggesting proton co-transport. MeJA was found to promote both its own transport and that of recent photoassimilate within 60 min. Furthermore, we found that MeJA can counter the inhibitory effect of the uncoupling agent, CCCP, on sugar transport, suggesting that MeJA affects the plasma membrane proton gradient. We also found that MeJA’s action may extend to the sucrose transporter, since MeJA countered the inhibitory effects of the sulfhydryl reagent, PCMBS, on the transport of photoassimilate.     

Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana

Plant viruses must enter the host vascular system in order to invade the young growing parts of the plant rapidly. Functional entry sites into the leaf vascular system for rapid systemic infection have not been determined for any plant/virus system. Tobacco mosaic virus (TMV) entry into minor, major and transport veins from non-vascular cells of Nicotiana benthamiana in source tissue and its exit from veins in sink tissue was studied using a modified virus expressing green fluorescent protein (GFP). Using a surgical procedure that isolated specific leaf and stem tissues from complicating vascular tissues, we determined that TMV could enter minor, major or transport veins directly from non-vascular cells to produce a systemic infection. TMV first accumulated in abaxial or external phloem-associated cells in major veins and petioles of the inoculated leaf and stems below the inoculated leaf. It also initially accumulated exclusively in internal or adaxial phloem-associated cells in stems above the inoculated leaf and petioles or major veins of sink leaves. This work shows the functional equivalence of vein classes in source leaves for entry of TMV, and the lack of equivalence of vein classes in sink leaves for exit of TMV. Thus, the specialization of major veins for transport rather than loading of photoassimilates in source tissue does not preclude virus entry. During transport, the virus initially accumulates in specific vascular-associated cells, indicating that virus accumulation in this tissue is highly regulated. These findings have important implications for studies on the identification of symplasmic domains and host macromolecule vascular transport.     

STUDIES ON THE LEAF OF AMARANTHUS RETROFLEXUS (AMARANTHACEAE): QUANTITATIVE ASPECTS,AND SOLUTE CONCENTRATION IN THE PHLOEM

The vascular system of the leaf of Amaranthus retroflexus L. was examined quantitatively, and plasmolytic studies were carried out on it to determine the solute concentration in cells of the phloem at various locations in the leaf. The proportion of phloem occupied by sieve tubes varies considerably with vein size and leaf size. Collectively, the cross-sectional area of sieve tubes of all tributaries at their points of entry into either a secondary or midvein far exceeds the total cross-sectional area of sieve tubes at the bases of those major veins. In addition, the total volume of sieve tubes in the “catchment area” of a secondary vein is much greater than total sieve-tube volume of the secondary vein itself. The plasmolytic studies revealed the presence of positive concentration gradients in the sieve tubes of the lamina from the minor veins and tips of the secondaries to the bases of the secondaries and from the tip to the base of the midvein. The C₅₀ (the estimated mannitol concentration plasmolyzing, on the average, 50% of the sieve-tube members) was 1.5 m for minor veins and tips of secondary veins and 1.1 m for the bases of secondaries; 1.3 m for the tip of the midvein and 0.6-0.7 m for the midvein in the basal third of the lamina.     

Characteristics of electrical signals in poplar and responses in photosynthesis

To gain an understanding of the role of electrical signaling in trees, poplar (Populus trichocarpa, Populus tremula x P. tremuloides) shoots were stimulated by chilling as well as flaming. Two kinds of signal propagation were detected by microelectrode measurements (aphid technique) in the phloem of leaf veins: (1) basipetal, short-distance signaling that led to rapid membrane hyperpolarization caused by K+-efflux within the leaf lamina; and (2) acropetal, long-distance signaling that triggered depolarization of the membrane potential in the leaf phloem. In the latter, the depolarizing signals travel across the stem from the manipulated leaves to adjacent leaves where the net CO2 uptake rate is temporarily depressed toward compensation. With regard to photosystem II, both heat-induced long-distance and short-distance signaling were investigated using two-dimensional "imaging" analysis of chlorophyll fluorescence. Both types of signaling significantly reduced the quantum yield of electron transport through photosystem II. Imaging analysis revealed that the signal that causes yield reduction spreads through the leaf lamina. Coldblocking of the stem proved that the electrical signal transmission via the phloem becomes disrupted, causing the leaf gas exchange to remain unaffected. Calcium-deficient trees showed a marked contrast inasmuch as the amplitude of the electrical signal was distinctly reduced, concomitant with the absence of a significant response in leaf gas exchange upon flame wounding. In summary, the above results led us to conclude that calcium as well as potassium is involved in the propagation of phloem-transmitted electrical signals that evoke specific responses in the photosynthesis of leaves.     

Translocation pathways in the petioles and stem between source and sink leaves of Populus deltoides Bartr. ex Marsh.

Microautoradiography was used to follow the translocation pathways of ¹⁴C-labeled photosynthate from mature source leaves, through the stem, to immature sink leaves three nodes above. Translocation occurred in specific bundles of the midveins and petioles of both the source and sink leaves and in the interjacent internodes. When each of six major veins in the lamina of an exporting leaf was independently spot-fed ¹⁴CO₂, label was exported through specific bundles in the petiole associated with that vein. When the whole lamina of a mature source leaf was fed ¹⁴CO₂, export occurred through all bundles of the lamina, but acropetal export in the stem was confined to bundles serving certain immature sink leaves. Cross-transfer occurred within the stem via phloem bridges. Leaves approaching maturity translocated photosynthate bidirectionally in adjacent subsidiary bundles of the petiole. That is, petiolar bundles serving the lamina apex were exporting unlabeled photosynthate while those serving the lamina base were simultaneously importing labeled photosynthate. The petioles and midveins of maturing leaves were strong sinks for photosynthate, which was diverted from the export front to differentiating structural tissues. The data support the idea of bidirectional transport in adjacent bundles of the petiole and possibly in adjacent sieve tubes within an individual bundle.Abbreviations C central leaf trace - L left leaf trace - LPI leaf plastochron index - R right leaf trace     

Phloem unloading of potato virus X movement proteins is regulated by virus and host factors

To determine the requirements for viral proteins exiting the phloem, transgenic plants expressing green fluorescent protein (GFP) fused to the Potato virus X (PVX) triple gene block (TGB)p1 and coat protein (CP) genes were prepared. The fused genes were transgenically expressed from the companion cell (CC)-specific Commelina yellow mottle virus (CoYMV) promoter. Transgenic plants were selected for evidence of GFP fluorescence in CC and sieve elements (SE) and proteins were determined to be phloem mobile based on their ability to translocate across a graft union into nontransgenic scions. Petioles and leaves were analyzed to determine the requirements for phloem unloading of the fluorescence proteins. In petioles, fluorescence spread throughout the photosynthetic vascular cells (chlorenchyma) but did not move into the cortex, indicating a specific barrier to proteins exiting the vasculature. In leaves, fluorescence was mainly restricted to the veins. However, in virus-infected plants or leaves treated with a cocktail of proteasome inhibitors, fluorescence spread into leaf mesophyll cells. These data indicate that PVX contributes factors which enable specific unloading of cognate viral proteins and that proteolysis may play a role in limiting proteins in the phloem and surrounding chlorenchyma.     

Sink to Source Transition of Populus Leaves

Development of the Populus leaf is presented as a model system to illustrate the sequence of events that occur during the sink to source transition. A Populus leaf is served by three leaf traces, each of which consists of an original procambial trace bundle that differentiates acropetally and continuously from more mature procambium in the stem and a complement of subsidiary bundles that differentiates bidirectionally from a leaf basal meristem. During development these subsidiary bundles maintain continuity through the meristematic region of the node. The basipetally developing subsidiary bunles form phloem bridges that serve to integrate adjacent leaf traces of the stem vasculature. Distal to the node the acropetally developing bundles from all three leaf traces are reoriented in a precise and orderly sequence to form tiers of petiolar bundles. These tiers of bundles extend into the midrib where bundles diverge at intervals as the major lateral veins. The dorsal-most tier of bundles extends to the lamina tip and each successive tier of bundles contributes to lateral veins situated more proximally in the lamina. Although the midrib and the major vein system differentiate acropetally in the lamina, they mature basipetally. Maturation of the mesophyll and other lamina tissues also mature basipetally. As a consequence of the basi-petal maturation process, the lamina tip matures very early and begins exporting photosynthates while the lamina base is still importing from other leaves. The transition of a leaf from sink to source status must therefore be considered as a progression of structural and functional events that occur in synchrony.     

Towards the proteome of Brassica napus phloem sap

The soluble proteins in sieve tube exudate from Brassica napus plants were systematically analyzed by 1-DE and high-resolution 2-DE, partial amino acid sequence determination by MS/MS, followed by database searches. 140 proteins could be identified by their high similarity to database sequences (135 from 2-DE, 5 additional from 1-DE). Most analyzed spots led to successful protein identifications, demonstrating that Brassica napus, a close relative of Arabidopsis thaliana, is a highly suitable model plant for phloem research. None of the identified proteins was formerly known to be present in Brassica napus phloem, but several proteins have been described in phloem sap of other species. The data, which is discussed with respect to possible physiological importance of the proteins in the phloem, further confirms and substantially extends earlier findings and uncovers the presence of new protein functions in the vascular system. For example, we found several formerly unknown phloem proteins that are potentially involved in signal generation and transport, e.g., proteins mediating calcium and G-protein signaling, a set of RNA-binding proteins, and FLOWERING LOCUS T (FT) and its twin sister that might be key components for the regulation of flowering time.     

Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.     

Salivary secretions by aphids interacting with proteins of phloem wound responses

Successful phloem feeding requires overcoming a number of phloem-related plant properties and reactions. The most important hurdle is formed by the phloem wound responses, such as coagulating proteins in the phloem sieve elements of the plant and in the capillary food canal in the insect's mouth parts, i.e. the stylets. It seems that in order to prevent protein clogging inside a sieve element, ejection of watery saliva plays an important role. This ejection is detected in the electrical penetration graph (EPG) as E1 salivation and always precedes phloem sap ingestion. During this feeding from sieve elements, another regular and concurrent salivation also occurs, the watery E2 salivation. This E2 saliva is added to the ingested sap and, it probably prevents phloem proteins from clogging inside the capillary food canal. Whatever the biochemical mode of action of the inhibition of protein coagulation might be, in some plants aphids do not seem to be able to prevent clogging, which may explain the resistance to aphids in these plants. The relevance of this hypothesis is demonstrated by new experimental results and is related to new EPG results from plants with phloem-located resistance.     

Photoassimilate-transport characteristics of nonchlorophyllous and green tissue in variegated leaves of Coleus blumei Benth

The nonchlorophyllous (albino) tissue of mature C. blumei leaves is a sink for photoassimilate. Transport from the green to the albino region of the same leaf was inhibited by cold and anoxia. When the green tissue of mature leaves was removed, the remaining albino portion imported labeled translocate from other mature leaves in the phloem. Photoassimilate unloading in the albino region of mature leaves was studied by quantitative autoradiography. The unloading was inhibited by cold but not by anoxia. No labeled photoassimilate could be detected in the free space of mature albino tissue by compartmental efflux analysis as phloem unloading proceeded in a N₂ atmosphere, indicating that unloading, may occur by a symplastic pathway as it apparently does in sink leaves of other species. The minor veins of mature albino leaf tissue did not accumulate exogenous [¹⁴C]sucrose. Minor veins of green tissue in the same leaves accumulated [¹⁴C]sucrose but, in contrast to other species studied to date, this accumulation was insensitive to the inhibitor p-chloromercuribenzensulfonic acid (PCMBS).In its capacity to import and unload photoassimilate, and in the inability, of the minor veins to accumulate exogenous sucrose, the albino region of the mature C. blumei lamina differs from mature albino tobacco leaves and darkened mature leaves of other species. This, together with evidence indicating that phloem loading in C. blumei and other species may occur by different routes and with different sensitivity to PCMBS, indicates that the mechanism of transfer of photoassimilates between veins and surrounding tissues, and the mechanism of the sink-source transition, may not be the same in the leaves of all species. It is speculated that the unusual properties of the C. blumei leaf may be a consequence of the presence, in the minor veins, of intermediary cells, large companion cells connected to the bundle sheath by abundant plasmodesmata.Abbreviation PCMBS p-chloromercuribenzenesulfonic acid