Phloem Loading in Coleus blumei in the Absence of Carrier-Mediated Uptake of Export Sugar from the Apoplast

Phloem loading in Coleus blumei Benth. leaves cannot be explained by carrier-mediated transport of export sugar from the apoplast into the sieve element-companion cell complex, the mechanism by which sucrose is thought to load in other species that have been studied in detail. Uptake profiles of the export sugars sucrose, raffinose, and stachyose into leaf discs were composed of two components, one saturable and the other not. Saturable (carrier-mediated) uptake of all three sugars was almost completely eliminated by the inhibitor p-chloromercuribenzenesulfonic acid (PCMBS). However, when PCMBS was introduced by transpiration into mature leaves it did not prevent accumulation of ¹⁴C-photosynthate in minor veins or translocation of labeled photosynthate from green to nonchlorophyllous regions of the leaf following exposure to ¹⁴CO₂. The efficacy of introducing inhibitor solutions in the transpiration stream was proven by observing saffranin O and calcofluor white movement in the minor veins and leaf apoplast. PCMBS introduced by transpiration completely inhibited phloem loading in tobacco leaves. Phloem loading in C. blumei was also studied in plasmolysis experiments. The carbohydrate content of leaves was lowered by keeping plants in the dark and then increased by exposing them to light. The solute level of intermediary cells increased in the light (phloem loading) in both PCMBS-treated and control tissues. A mechanism of symplastic phloem loading is proposed for species that translocate the raffinose series of oligosaccharides.     

Phloem Unloading in Developing Leaves of Sugar Beet : II. Termination of Phloem Unloading

Phloem unloading in developing leaves of Beta vulgaris L. (`Klein E' multigerm) occurred from successively higher order branches of veins as leaves matured. Phloem unloading was studied in autoradiographs of leaf samples taken at various times during the arrival of a pulse of ¹⁴C-labeled photoassimilate. Extension of mass flow of sieve element contents into leaf vein branches was determined from the high level of radiolabel in veins soon after first arrival of the pulse. Rapid entry, indicative of mass flow through open sieve pores, occurred down to the fourth division of veins in young, importing leaves and to the fifth or terminal branch in importing regions near the zone of transition from sink to source. The rate of unloading decreased with leaf age, as evidenced by the increased time required for the vein-mesophyll demarcation to become obscured. The rate of import per unit leaf area, measured by steady state labeling with ¹⁴CO₂ also decreased as a leaf matured. The decline in import appeared to result from progressive changes that increased resistance to unloading of sieve elements and eventually terminated phloem unloading.     

Source-sink coupling in young barley plants and control of phloem loading

Phloem loading of carbohydrate within a mature exporting leaf of a barley seedling is shown to respond quickly to a change in the temperature of the root and the shoot meristem. This is interpreted as a close coupling between source supply and sink demand for carbohydrate, through the hydrostatic pressure gradient linking source and sink generated by the solute concentration within the sieve tubes. This interpretation was tested by using anoxia to alter solute concentration within the sieve tubes of one region of a leaf while observing phloem loading in an adjacent region. Responses to anoxia could not be explained by the above model, suggesting that either this model is incorrect or other signalling pathways are involved. There is evidence in the literature for coarse control of phloem loading but no evidence was found of fine control by solute content of the loaded sieve elements.     

The Effects of Fusicoccin,Abscisic Acid,and Benzyladenine on the Transport and Partitioning of Substances as Related to Cytokinin Sink Activity

We tested the possible cytokinin effect on the functioning of the active transport system involved in the assimilate loading into the phloem as a cause for the cytokinin sink and retention effect. This effect is manifested in the deceleration of substance export from and the stimulation of substance import to the sites of local phytohormone application to the mature detached leaf from untreated leaf areas. To affect the membrane mechanisms of the substance transport, we used leaf treatment with the phytotoxin fusicoccin, an enhancer of plasmalemmal H⁺-ATPase and a potential stimulator of assimilates export, and with the phytohormone ABA affecting transport, metabolism, and plant growth. However, fusicoccin did not enhance ¹⁴C-sucrose export from the leaf blade and did not interfere with the cytokinin-induced export deceleration. ABA reduced substantially ¹⁴C export from the leaf but eliminated the cytokinin effect on this process. Similar results were obtained for broad bean (Vicia faba L.) leaves with apoplastic phloem loading, involving H⁺-ATPase activity, and pumpkin (Cucurbita pepo L.) leaves with symplastic phloem loading, that is, occurring without sucrose transmembrane translocation and without H⁺-ATPase involvement. The conclusion is that the cytokinin-induced development of sink zones in source leaves is not related to the membrane mechanisms of the substance transport in the mesophyll–phloem system. The data obtained support the idea that the cause for the cytokinin sink and retention effect is the enhancement of elongation growth and total activation of metabolism in the mesophyll cells of the detached leaf.     

Phloem Loading of Sucrose: pH Dependence and Selectivity

Autoradiographic, plasmolysis, and ¹⁴C-metabolite distribution studies indicate that the majority of exogenously supplied ¹⁴C-sucrose enters the phloem directly from the apoplast in source leaf discs of Beta vulgaris. Phloem loading of sucrose is pH-dependent, being markedly inhibited at an apoplast pH of 8 compared to pH 5. Kinetic analyses indicate that the apparent K_m of the loading process increases at the alkaline pH while the maximum velocity, V_max, is pH-independent. The pH dependence of sucrose loading into source leaf discs translates to phloem loading in and translocation of sucrose from intact source leaves. Studies using asymmetrically labeled sucrose ¹⁴C-fructosyl-sucrose, show that sucrose is accumulated intact from the apoplast and not hydrolyzed to its hexose moieties by invertase prior to uptake. The results are discussed in terms of sucrose loading being coupled to the co-transport of protons (and membrane potential) in a manner consistent with the chemiosmotic hypothesis of nonelectrolyte transport.     

Phloem Pressure Differences and C-Assimilate Translocation in Ecballium elaterium

The role of phloem turgor pressure in ¹⁴C-assimilate translocation in Ecballium elaterium A. Rich was studied. The direction of translocation was manipulated by two methods: darkening, or defoliation, of the upper or lower halves of the shoots. After 24 hours of labeled assimilate movement, sieve tube turgor levels were measured with the phloem needle technique. Distribution of label, determined by autoradiography and counting, revealed a direct correlation between the direction of assimilate transport and the pressure difference. Phloem turgor levels always decreased in the stem of darkened shoots; this resulted in greater pressure differences in the stem between the source leaf receiving ¹⁴CO₂ and treated regions.     

Sink Effect of Cytokinin in Detached Leaves Is Not Caused by Structural Rearrangements of Phloem

The sink effect of cytokinin is manifested as a decrease in source capacity and the induction of sink activity in the phytohormone-treated region of a mature excised leaf. In order to find out whether this effect was due to the direct action of cytokinin on the phloem structure, two types of phloem terminals were examined. In pumpkin (Cucurbita pepo L.) leaves, the phloem terminals are open; i.e., they are linked to mesophyll by numerous symplastic connections, which are located in narrow areas called plasmodesmal pit fields. In broad bean (Vicia faba L.) leaves, the phloem terminals belong to the closed type and have no symplastic links with mesophyll. The electron microscopic study of terminal phloem did not reveal any structural changes in the companion cells, which could account for the suppression of assimilate export. The treatment of leaves with cytokinin neither disturbed the structure of plasmodesmal pit fields in pumpkin leaves nor eliminated the wall protuberances (the ingrowths promoting phloem loading) in bean leaves. No evidence was obtained that the cytokinin-induced import of assimilates in mature leaves is caused by the recovery of meristematic activity, i.e., by either formation of new phloem terminals having immature sieve elements capable of unloading or by the development of new sieve elements within the existing veins. Cytokinin did not induce de novo formation of phloem elements. Structural characteristics of the leaf phloem, such as the number of branching orders in the venation pattern, the number of vein endings per areole, the number of areoles per leaf, the area of one areole, and the number of sieve elements per bundle remained unaltered. It is concluded that the sink effect of cytokinin in excised leaves cannot be determined by alteration of the phloem structure.     

Distorted phytochrome action spectra in green plants

An evaluation was made of the extent which a Münch-type pressure flow mechanism (i.e., osmotically-generated pressure flow) might contribute to phloem transport in soybean. Estimates of sucrose concentrations in source (leaf) and sink (root) sieve tubes were obtained by a negativestaining procedure. Water potential measurements of the leaf and of the nutrient solution allowed calculation of the turgor pressures in source and sink sieve tubes. The turgor difference between source and sink sieve tubes was compared to that required to drive translocation at the observed velocity between the source and sink, as measured by [¹⁴C] photosynthate movement. Sieve-tube conductivity was calculated from the sieve-tube dimensions, assuming an essentially unobstructed pathway. In three experiments, the sucrose concentration was consistently higher in source sieve tubes (an average of 11.5%) than in sink sieve tubes (an average of 5.3%). The ratio of these values (2.3:1) agreed reasonably well with an earlier ratio for source/sink sieve tube concentrations of 1.8:1, obtained by quantitative microautoradiography. The resulting calculated turgor difference (an average of 4.1 bars) was adequate to drive a pressure flow mechanism at the observed translocation velocities (calculated to require a turgor difference of 1.2 to 4.6 bars). No other force need be presumed to be involved.This work was presented in part at a joint U.S.-Australian Conference on Transport and Transfer Processes in Plants, Canberra, Australia, December 15–20, 1975; see Fisher (1976)     

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 in tobacco sink leaves: insensitivity to anoxia indicates a symplastic pathway

Phloem unloading in transition sink leaves of tobacco (Nicotiana tabacum L.) was analyzed by quantitative autoradiography. Detectable levels of labeled photoassimilates entered sink leaves approx. 1 h after source leaves were provided with ¹⁴CO₂. Samples of tissue were removed from sink leaves when label was first detected and further samples were taken at the end of an experimental phloem-unloading period. The amount of label in veins and in surrounding cells was determined by microdensitometry of autoradiographs using a microspectrophotometer. Photoassimilate unloaded from first-, second-and third-order veins but not from smaller veins. Import termination in individual veins was gradual. Import by the sink leaf was completely inhibited by exposing the sink leaf to anaerobic conditions, by placing the entire plant in the cold, or by steam-girdling the sink-leaf petiole. Phloem unloading was completely inhibited by cold; however, phloem unloading continued when the sink-leaf petiole was steam girdled or when the sink leaf was exposed to a N₂ atmosphere. Compartmental efflux-analysis indicated that only a small percentage of labeled nutrients was present in the free space after unloading from sink-leaf veins in a N₂ atmosphere. The results are consistent with passive symplastic transfer of photoassimilates from phloem to surrounding cells.Symbol VI radio of ¹⁴C in veins and interveinal tissue     

Dissimilar phloem loading in leaves with symplasmic or apoplasmic minor-vein configurations

Plant species were selected on the basis of abundant or no symplasmic continuity between sieveelement-companion-cell (SE-CC) complexes and adjacent cells in the minor veins. Symplasmic continuity and discontinuity are denoted, respectively, as symplasmic and apoplasmic minor-vein configurations. Discs of predarkened leaves from which the lower epidermis had been removed, were exposed to ¹⁴CO₂. After 2 h of subsequent incubation, phloem loading in control discs and discs treated with p-chloromercuribenzenesulfonic acid (PCMBS) was recorded by autoradiography. Phloem loading was strongly suppressed by PCMBS in minor veins with symplasmically isolated SE-CC complexes (Centaurea, Impatiens, Ligularia, Pelargonium, Pisum, Symphytum). No significant inhibition of phloem loading by PCMBS was observed in minor veins containing sieve elements with abundant symplasmic connections (Epilobium, Fuchsia, Hydrangea, Oenothera, Origanum, Stachys). Phloem loading in minor veins with both types of SE-CC complex (Acanthus) had apoplasmic features. The results provide strong evidence for coincidence between the mode of phloem loading and the minor-vein configuration. The widespread occurrence of a symplasmic mode of phloem loading is postulated.Abbreviations PCMBS p-chloromercuribenzenesulfonic acid - SE-CC complex sieve-element-companion-cell complex     

The biosynthesis and translocation of 14C-IAA in Ricinus communis

The biosynthesis of ¹⁴C-IAA from ¹⁴C-tryptophan applied to abraded leaves of Ricinus communis and its subsequent export through the phloem were studied. Phloem sap was collected at intervals from incisions made in the stem below the IAA fed leaf. Any upward movement of label through the phloem or downward movement of phloem mobile compounds from leaves above the treated one were restricted by bark-ringing the plants.TLC and HPLC analyses of the collected sap indicate that some conversion of ¹⁴C-tryptophan to ¹⁴C-IAA had occurred. Subsequent GC-MS analysis of the HPLC purified samples of phloem sap revealed high levels of endogenous IAA transported from the fed leaf. The high ratio of unlabelled/labelled IAA in the phloem sap makes unequivocal confirmation by GC-MS of the predicted biosynthesis of ¹⁴C-IAA impossible. It is postulated that IAA is synthesised from tryptophan in mature leaves and exported to developing sink tissues with the flow of photoassimilates in the phloem.     

A three-step screening procedure to identify the mode of phloem loading in intact leaves

A three-step screening method was developed to identify the mode of phloem loading in intact leaves. Phloem loading of ¹⁴CO₂-derived photosynthate was challenged by p-chloromercuribenzenesulfonic acid (PCMBS) in leaves of dicotyledons with either a symplasmic (type 1, with intermediary cells as companion cells) or apoplasmic (type 2b, with transfer cells as companion cells) minor-vein configuration. Firstly, photosynthate export as the result of phloem loading was measured by collection of phloem exudate from the petiole. The PCMBS had virtually no effect on photosynthate export in representatives of type-1 families (Lamiaceae, Lythraceae, Onagraceae, Saxifragaceae). In contrast, photosynthate export was strongly reduced by PCMBS in representatives of type-2b families (Asteraceae, Balsaminaceae, Dipsacaceae, Linaceae, Tropaeolaceae, Valerianaceae) and type-2b members of polytypical families (Fabaceae, Scrophulariaceae). Secondly, densitometric measurements of leaf autoradiographs demonstrated that the contrast between the mesophyll and the lower-order veins was hardly affected by PCMBS treatment in type-1 species, whereas PCMBS strongly reduced the contrast in type-2b species. Thirdly, separate ¹⁴C-radioassays of vein and mesophyll tissues confirmed this observation. The three-step procedure thus revealed a strong and consistent reduction of phloem loading by PCMBS in type-2b species which was absent in type-1 species. In conclusion, phloem loading in type-2b species occurs via the apoplast and type-1 species execute an alternative — most likely symplasmic — mode of phloem loading.Abbreviations PCMBS p-chloromercuribenzenesulfonic acid - SE/CC-complex sieve element/companion cell complex We gratefully acknowledge the expert help of Dr. Maarten Terlou, Department of Image Processing and Design, University of Utrecht, in carrying out the densitometric measurements.     

Effect of Exogenously Supplied Foliar Potassium on Phloem Loading in Beta vulgaris L

The effect of foliar application of K⁺ on processes associated with phloem loading was investigated in source leaves of sugar beet (Beta vulgaris L.). KCI was supplied exogenously at concentrations of up to 100 millimolar in the solution bathing the abraded upper epidermis of source leaves. K⁺ added at concentrations below 30 millimolar generally promoted the rate of export of material derived from ¹⁴CO₂ but not from exogenously applied [¹⁴C]sucrose. Paralleling promotion of export, the level of material derived from photosynthesis, which was released into the bathing solution, also increased in response to addition of K⁺ to the free space. Net photosynthetic rate was not affected. K⁺ at 5 and 15 millimolar concentrations did not stimulate uptake of [¹⁴C]sucrose into source leaf discs.     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

In vitro and in vivo modification of sugar transport and translocation in celery by phytohormones

In an attempt to address the controversy in the literature as to whether phytohormones have any direct effect on phloem loading of sucrose, we investigated the effect of gibberellic acid (GA₃) and indoleacetic acid (IAA) on sugar transport and translocation in celery (Apium graveolens L. cv. Utah 5270). Both hormones enhanced sucrose uptake into isolated vascular bundles and phloem tissue of celery and enhanced the export of ¹⁴C assimilates from leaves of intact plants in vivo. The hormone-induced increase of uptake into isolated vascular bundles or phloem was specific for sucrose and mannitol which are translocated in phloem. Furthermore, the hormone-induced increase in translocation was not due to an increase in sink demand, since neither glucose nor sucrose uptake rates were affected in the storage parenchyma tissue discs (sink region) in the presence of GA₃ or IAA. The evidence suggests that phytohormones may have a direct effect on phloem loading of sucrose. The possibility of short-term GA₃ and IAA effects on processes resulting in membrane transport of sugars in celery is discussed.     

<Emphasis Type="Italic">Amborella trichopoda</Emphasis>, plasmodesmata,and the evolution of phloem loading

Phloem loading is the process by which photoassimilates synthesized in the mesophyll cells of leaves enter the sieve elements and companion cells of minor veins in preparation for long distance transport to sink organs. Three loading strategies have been described: active loading from the apoplast, passive loading via the symplast, and passive symplastic transfer followed by polymer trapping of raffinose and stachyose. We studied phloem loading in Amborella trichopoda, a premontane shrub that may be sister to all other flowering plants. The minor veins of A. trichopoda contain intermediary cells, indicative of the polymer trap mechanism, forming an arc on the abaxial side and subtending a cluster of ordinary companion cells in the interior of the veins. Intermediary cells are linked to bundle sheath cells by highly abundant plasmodesmata whereas ordinary companion cells have few plasmodesmata, characteristic of phloem that loads from the apoplast. Intermediary cells, ordinary companion cells, and sieve elements form symplastically connected complexes. Leaves provided with ¹⁴CO₂ translocate radiolabeled sucrose, raffinose, and stachyose. Therefore, structural and physiological evidence suggests that both apoplastic and polymer trapping mechanisms of phloem loading operate in A. trichopoda. The evolution of phloem loading strategies is complex and may be difficult to resolve.     

L-Ascorbic acid is accumulated in source leaf phloem and transported to sink tissues in plants

L-Ascorbic acid (AsA) was found to be loaded into phloem of source leaves and transported to sink tissues. When L-[(14)C]AsA was applied to leaves of intact plants of three different species, autoradiographs and HPLC analysis demonstrated that AsA was accumulated into phloem and transported to root tips, shoots, and floral organs, but not to mature leaves. AsA was also directly detected in Arabidopsis sieve tube sap collected from an English green aphid (Sitobion avenae) stylet. Feeding a single leaf of intact Arabidopsis or Medicago sativa with 10 or 20 mM L-galactono-1,4-lactone (GAL-L), the immediate precursor of AsA, lead to a 7- to 8-fold increase in AsA in the treated leaf and a 2- to 3-fold increase of AsA in untreated sink tissues of the same plant. The amount of AsA produced in treated leaves and accumulated in sink tissues was proportional to the amount of GAL-L applied. Studies of the ability of organs to produce AsA from GAL-L showed mature leaves have a 3- to 10-fold higher biosynthetic capacity and much lower AsA turnover rate than sink tissues. The results indicate AsA transporters reside in the phloem, and that AsA translocation is likely required to meet AsA demands of rapidly growing non-photosynthetic tissues. This study also demonstrates that source leaf AsA biosynthesis is limited by substrate availability rather than biosynthetic capacity, and sink AsA levels may be limited to some extent by source production. Phloem translocation of AsA may be one factor regulating sink development because AsA is critical to cell division/growth.     

Enhancement of [C]Sucrose Export from Source Leaves of Vicia faba by Gibberellic Acid

The effect of gibberellic acid (GA₃) on sucrose export from source leaves was studied in broad bean (Vicia faba L.) plants trimmed of all but one source and one sink leaf. GA₃ (10 micromolar) applied to the source leaf, enhanced export of [¹⁴C]sucrose (generated by ¹⁴CO₂ fixation) to the root and to the sink leaf. Enhanced export was observed with GA treatments as short as 35 minutes. When GA₃ was applied 24 hours prior to the ¹⁴CO₂ pulse, the enhancement of sucrose transport toward the root was abolished but transport toward the upper sink leaf was unchanged. The enhanced sucrose export was not due to increased photosynthetic rate or to changes in the starch/sucrose ratio within the source leaf; rather, GA₃ increased the proportion of sucrose exported. After a 10-min exposure to [¹⁴C]GA₃, radioactivity was found only in the source leaf. Following a 2 hour exposure to [¹⁴C]GA₃, radioactivity was distributed along the entire stem and was present in both the roots and sink leaf. Extraction and partitioning of GA metabolites by thin layer chromatography indicated that there was a decline in [¹⁴C]GA₃ in the lower stem and root, but not in the upper stem. This pattern of metabolism is consistent with the disappearance of the GA₃ effect in the lower stem with time after treatment. We conclude that in the short term, GA₃ enhances assimilate export from source leaves by increasing phloem loading. In the long term (24 hours), the effect of GA₃ is outside the source leaf. GA₃ accumulates in the apical region resulting in enhanced growth and thus greater sink strength. Conversely, GA₃ is rapidly metabolized in the lower stem thus attenuating any GA effect.