Apoplastic Phloem Unloading in the Stem of Bean

Sucrose has been found in the apoplast of bean stems at a concentrationof 25–60 mM with an axial concentration gradient in theappropriate direction for Munch translocation. Removal of theepidermis from a 50 mm length of stem enabled the washout oflabelled photosynthate from the apoplast. The rate of labelwashout was strongly dependent on temperature, and the rateincreased on blockage of phloem pathways to the main sink forthat assimilate. Washout did not reduce when the bathed tissuewas plasmolyzed. We propose that sucrose is unloaded from thephloem into the apoplast, and a sucrose concentration is maintainedthere by a balance of sucrose uptake into sink tissue or reloadinginto the phloem. It is proposed that the apoplastic pool ofphotosynthate can act to buffer sudden changes in phloem contentswhen there are rapid changes in source-sink configuration. Key words: Sucrose, Phaseolus vulgaris, Apoplast, Phloem unloading     

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.     

Measurement of Unloading and Reloading of Photo-assimilate within the Stem of Bean

There is growing evidence that the phloem transport pathwayis leaky with passive unloading into the apoplast and activereloading from the apoplast. In this paper we report in vivomeasurements of unloading and reloading in immature stems ofbean expressed as a percentage per unit length of the photo-assimilateflow within the transport pathway. Gross unloading was up to6% cm^-1 and net unloading up to 4% cm^-1. Time constants of bulkflow through 10 cm of stem were about 4-6 min whereas thoseof net unloading were 8-13 min and of gross unloading, at themost, 7-8 min. Key words: Phloem transport, apoplast, bean stem     

Water Flow Across the Sieve-Tube Boundary: Estimating Turgor and Some Implications for Phloem Loading and Unloading. II. Phloem in the Stem

Confirming a previous analysis by Lang (1974), it is concludedthat in tree trunks, phloem turgor and turgor gradients maybe estimated from osmotic pressure and osmotic-pressure gradients,respectively. The present analysis is an improvement becauseit is based on observed osmotic-pressure gradients rather thansupposed turgor gradients, and allowance is made for sucroseunloading and gradients of external water potential. It is concludedthat the rate of sucrose unloading in tree trunks must be lessthan 50 nmol m^–2 S m^–1. In small plants, higherrates of unloading (100 nmol m m^–2 S m^–1) and steeperconcentration gradients will lead to larger errors, but turgorpressures can still be estimated with acceptable accuracy. Oneshould be more cautious when considering turgor gradients insmall plants, although it seems likely that reasonable estimateswill still be obtained. Assuming plasmodesmatal transport throughan unconstricted cytoplasmic annulus, it is concluded that thesieve elements and their associated cells will sustain verysimilar turgor and osmotic pressures. Convection and diffusioncan both contribute significantly to plasmodesmatal sucroseunloading. Similarly, the plasmodesmatal volume flux will reflecta combination of pressure flow and osmosis. Water fluxes acrossthe sieve element plasmalemma and through the plasmodesmatacan be in opposite directions. It may be possible to assessthe extent of hydraulic coupling between the sieve elementsand their associated cells from studies of phloem water relations Phloem, turgor, osmotic pressure, plasmodesmata, phloem unloading, Munch hypothesis     

Phloem Unloading in Developing Leaves of Sugar Beet : I. Evidence for Pathway through the Symplast

Physiological and transport data are presented in support of a symplastic pathway of phloem unloading in importing leaves of Beta vulgaris L. (`Klein E multigerm'). The sulfhydryl reagent p-chloromercuribenzene sulfonic acid (PCMBS) at concentration of 10 millimolar inhibited uptake of exogenous [¹⁴C]sucrose by sink leaf tissue over sucrose concentrations of 0.1 to 5.0 millimolar. Inhibited uptake was 24% of controls. The same PCMBS treatment did not affect import of ¹⁴C-label into sink leaves during steady state labeling of a source leaf with ¹⁴CO₂. Lack of inhibition of import implies that sucrose did not pass through the free space during unloading. A passively transported xenobiotic sugar, l-[¹⁴C]glucose, imported by a sink leaf through the phloem, was evenly distributed throughout the leaf as seen by whole-leaf autoradiography. In contrast, l-[¹⁴C]glucose supplied to the apoplast through the cut petiole or into a vein of a sink leaf collected mainly in the vicinity of the major veins with little entering the mesophyll. These patterns are best explained by transport through the symplast from phloem to mesophyll.     

Developmental Changes in the Anatomy of the Sugarcane Stem in Relation to Phloem Unloading and Sucrose Storage

Transverse sections of immature and mature sugarcane internodes were investigated anatomically with white and fluorescence light microscopy. The pattern of lignification and suberization was tested histo-chemically. Lignification began in the xylem of vascular bundles and progressed through the sclerenchymatic bundle sheath into the storage parenchyma. Suberization began in parenchyma cells adjacent to vascular bundle sheaths and spread to the storage parenchyma and outer sheath cells. In mature internodes most of the storage parenchyma was lignified and suberized to a significant degree, except in portions of walls of isolated cells. The pattern of increasing lignification and suberization in maturing internodes more or less paralleled an increase of sucrose in stem tissue. In mature internodes having a high sucrose concentration, the vascular tissue was surrounded by thick-walled, lignified and suberized sclerenchyma cells. The apoplastic tracer dyes triso-dium 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS) and amido black 10 B, fed into cut ends of the stalk, wereconfined to the vascular bundles in all internodes above the one that was cut — with no dye apparently in storage parenchyma tissue. Thus both structural and experimental evidence is consistent with vascular tissue being increasingly isolated from the storage parenchyma as maturation of the tissue proceeds. We conclude that in mature internodes the pathway for sugars from the phloem to the storage parenchyma is symplastic. The data suggest that an increasingly greater role for a symplastic pathway of sugar transfer occurs as the tissue undergoes lignification/suberization.     

Sucrose Transport and Phloem Unloading in Stem of Vicia faba: Possible Involvement of a Sucrose Carrier and Osmotic Regulation

Stems of Vicia faba plants were used to study phloem unloading because they are hollow and have a simple anatomical structure that facilitates access to the unloading site. After pulse labeling of a source leaf with ¹⁴CO₂, stem sections were cut and the efflux characteristics of ¹⁴C-labeled sugars into various buffered solutions were determined. Radiolabeled sucrose was shown to remain localized in the phloem and adjacent phloem parenchyma tissues after a 2-hour chase. Therefore, sucrose leakage from stem segments prepared following a 75-minute chase period was assumed to be characteristic of phloem unloading. The efflux of ¹⁴C assimilates from the phloem was enhanced by 1 millimolar p-chloromercuribenzene sulfonic acid (PCMBS) and by 5 micromolar carbonyl cyanide m-chlorophenly hydrazone (CCCP). However, PCMBS inhibited and CCCP enhanced general leakage of nonradioactive sugars from the stem segments. Sucrose at concentrations of 50 millimolar in the free space increased efflux of [¹⁴C]sucrose, presumably through an exchange mechanism. This exchange was inhibited by PCMBS and abolished by 0.2 molar mannitol. Increasing the osmotic concentration of the efflux medium with mannitol reduced [¹⁴C]sucrose efflux. However, this inhibition seems not to be specific to sucrose unloading since leakage of total sugars, nonlabeled sucrose, glucose, and amino acids from the bulk of the tissue was reduced in a similar manner. The data suggest that phloem unloading in cut stem segments is consistent with passive efflux of sucrose from the phloem to the apoplast and that sucrose exchange via a membrane carrier may be involved. This is consistent with the known conductive function of the stem tissues, and contrasts with the apparent nature and function of unloading in developing seeds.     

Abscisic Acid Movement into the Apoplastic solution of Water-Stressed Cotton Leaves: Role of Apoplastic pH

Leaves of cotton (Gossypium hirsutum L.) were subjected to overpressures in a pressure chamber, and the exuded sap was collected and analyzed. The exudate contained low concentrations of solutes that were abundant in total leaf extracts, and photosynthetic rates and stomatal conductance were completely unaffected by a cycle of pressurization and rehydration. These criteria and others indicate that the experimental techniques inflicted no damage upon the leaf cells. The pH and abscisic acid (ABA) content of the apoplastic fluid both increased greatly with pressure-induced dehydration. Although ABA concentrations did not reach a steady state, the peak levels were above 1 micromolar, an order of magnitude greater than bulk ABA concentrations of the leaf blades. Treatment of leaves with fusicoccin decreased the K⁺ concentration, greatly reduced the pH rise, and completely eliminated the increase in ABA in the apoplast upon dehydration. When water-stressed leaves were pressurized, the pH of the exuded sap was increased by 0.2 units per 1 megapascal decrease in initial leaf water potential. Buffer capacity of the sap was least in the pH range of interest (6.5-7.5), allowing extremely small changes in H⁺ fluxes to create large changes in apoplastic pH. The data indicate that dehydration causes large changes in apoplastic pH, perhaps by effects on ATPases; the altered pH then enhances the release of ABA from mesophyll cells into the apoplastic fluid.     

Anatomy of the Secondary Phloem and the Crystalliferous Phloem Fibers in the Stem of Torreya grandis

The structure of the secondary phloem and the development of the crystaleiferous phloem fibers in the stem of Torrey grandis were observed under the ligth microscope and SEM. The secondary phloem is composed of sieve cells, phloem parenchyma cells, crystalliferous phloem fibers and stone cells in the longitudinal system, and the uniserite homogeneous phloem rays consisting of parenchyma cells only in the radial system. In the cross section, there are 3–9 sieve cells in radial rows forming discontinuous tangential layers, the crystalliferous phloem fibers often in a single discontinuous tangential layer and the stone cells dispersed in rangential layer of phloem parenchyma. The developmental process of crystalliferous phloem fibers is as follows: initial cells appeared in the end of April and were well differentiated in the first week of May. Some crystals were deposited in the primary wall, while others were free in the cell. At the end of May, the secondary wall of most crysalliferous phloem fibers started to be thickened. With the thickening of the secondary wall, all the crystals were embedded in the wall from June to August From the end of September to the early days of October, the crystalliferous phloem fibers reached their full maturation. It is shown by microchemical identification and EDAX analysis that the crystals embedded in the wail of crystalliferous phloem fibers are calcium oxalate crystals.     

Phloem Unloading of Amino Acids at the Site of Attachment of Cuscuta europaea

By washing out ¹⁴C-solutes or ³H-solutes in 0.5 mm CaSO₄ during a period of 5 to 6 hours, the release of amino acids by excised stem segments of broad bean (Vicia faba L. cv Witkiem) was studied. Three hours after pulse labeling with l-valine, l-asparagine, or α-aminoisobutyric acid (AIB), hollow stem segments were excised from the plant and incubated in a washout solution.     

Evidence for a Flow of Water into Sieve Tubes Associated with Phloem Loading

Evidence that there is a lateral water flow associated withthe loading of sieve tubes is presented. The change in phloemtransport dynamics, measured using ¹¹C, induced by leaf anoxiais shown to be consistent with a reduction in lateral waterflow brought about by a decline in sucrose loading. The temporalprofiles of tracer activity are used to derive a simple holisticmodel of the phloem pathway which is able to account for theobserved dynamics.     

Phloem Mobility of Boron is Species Dependent: Evidence for Phloem Mobility in Sorbitol-rich Species

Boron is generally considered to be phloem immobile or to haveonly limited phloem mobility in higher plants. Evidence suggests,however, that B may be mobile in some species within thePyrus,Malus andPrunusgenera. These genera utilize sorbitol as a primarytranslocated photosynthate and it has been clearly demonstratedthat B forms stable complexes with sorbitolin vitro.In the researchpresented here we demonstrate, further, that B is freely phloemmobile inPyrus, MalusandPrunusspecies and suggest that thisis mediated by the formation and transport of B-sorbitol complexes. The pattern of B distribution within shoot organs and the translocationof foliar-applied, isotopically-enriched¹⁰B was studied in sixtree species. Results demonstrate that in species in which sorbitolis a major sugar (sorbitol-rich), B is freely mobile while inspecies that produce little or no sorbitol (sorbitol-poor) Bis largely immobile. The sorbitol-rich species used here werealmond [Prunus amygdalusB. syn.P. dulcis(Mill.)], apple (MalusdomesticaB.) and nectarine (Prunus persicaL. B. var.nectarinaM.),sorbitol-poor species included fig (Ficus caricaL.), pistachio(Pistacia veraL.) and walnut (Juglans regiaL.). In sorbitol-richspecies foliar applied¹⁰B was transported from the treated leavesto adjacent fruit and specifically to the fruit tissues (hull,shell or kernel) that developed during the experimental period.Whereas, foliar-applied¹⁰B was rapidly translocated out of leaves,only a small percentage of the¹¹B present in the leaf at thetime of foliar application was retranslocated. In sorbitol-richspecies, B concentrations differed only slightly between oldand young leaves while fruit tissue had significantly greaterB concentrations than leaves. In contrast, sorbitol-poor specieshad significantly higher B concentrations in older leaves thanyoung leaves while fruit tissue had the lowest B concentration.This occurred irrespective the source of plant B (soil, solutionor foliar-applied). In a subsequent experiment the growth ofapple trees in solutions free of applied B was maintained solelyby foliar applications of B to mature leaves. These resultsindicate that B is mobile in species that produce significantamounts of sorbitol. We propose that the mobility of B in thesespecies is mediated by the formation of B-sorbitol complexes. Almond; Prunus amygdalus ; apple; Malus domestica; nectarine; Prunus persica; fig; Ficus carica; pistachio; Pistacia vera; walnut; Juglans regia; boron; phloem mobility; deficiency; toxicity; inductively coupled plasma-mass; spectrometer     

Evidence for Symplastic Phloem Unloading with Concomitant High Activity of Acid Cell Wall Invertase in Agrobacterium tumefaciens-Induced Plant Tumors*

In stems of Ricinus communis and leaves of Kalanchoë daigremontiana, rapidly growing tumors were induced by the wild type strains of Agrobacterium tumefaciens C58 and A281 (p35 Sgusint). Transformed cells, monitored by histochemical β-glucuronidase (GUS) staining, showed GUS activity in K. daigremontiana tumors in up to 100% of the tissue. In R. communis tumors, however, GUS activity was patchy, probably due to interference in gus expression from highly active phenolic compounds. Functionality of the sieve elements within the vascular bundles of the tumor and their connection with host stem bundles were shown by applying fluorescein to source leaves as a tracer of phloem-mobile solutes. The transport pathway within the tumor and the mechanism of phloem unloading were investigated by iontophoretic injection of Lucifer yellow CH into sieve tubes. Apparent symplastic solute unloading into parenchyma cells was confirmed by localizing common primary pit fields by staining them with aniline blue. In spite of the evidence for symplastic unloading, the activity of acid cell wall invertase (CWI) was about tenfold higher in tumor than in the adjacent host stem tissue. These results indicate primary independence of phloem unloading of CWI in tumors.     

Water Flow Across the Sieve-Tube Boundary: Estimating Turgor and Some Implications for Phloem Loading and Unloading. III. Phloem in the Leaf

The theory described in Part I of this series is applied hereto the loading of water and sucrose into the sieve-element-companion-cell(se-cc) complexes of minor veins. It is concluded that symplasticphloem loading cannot account for large increases in osmoticpressure from mesophyll to se-cc complex or the dilution ofsieve tube sap which is evident in leaves. By contrast, loadingfrom the free space into a symplastically isolated se-cc complexcan account for both these observations. Within the se-cc complex,sucrose and water will be transported from the companion cellsto the sieve elements by a pressure-driven flow of solutionthrough the cytoplasmic annuli of plasmodesmata. The associatedchanges in turgor and osmotic pressure are small, and so these-cc complex can be regarded as a single compartment with respectto water potential. The assumption that this compartment isat water flux equilibrium will lead to significant overestimatesof turgor gradients. However, if the trans-membrane water potentialdifference and the external water potential are taken into account,the correlation of such gradients with sieve-element dimensionsand / or transport velocities provides one means of testingthe Munch hypothesis of phloem transport Phloem, turgor, osmotic pressure, plasmodesmata, Münch hypothesis, Phloem loading     

Comparative Anatomy of the Secondary Phloem of the Stem in Cephalotaxus

The family Cephalotaxaceae contains only one genus, Cephalotaxus, with 9 species and some varieties, mostly endemic to China. The present paper deals with the comparative anatomy of secondary phloem of stems in 4 species and I cultivar, of this genus under light microscope and scanning electron microscope. The main results are as follows: (1) The structure of secondary phloem is rather uniform in the various species of this genus. In cross section, sieve cells, phloem parenchyma cells form continuous tangential rows of one cell in width respectively, which occur alternately. Sclerenchyma cells also form continuous tangential rows, each with a radial width of 1-4 cells. The interval between the rows is rather wide. (2) The type of phloem fiber and the quantity of sclereids can be served as the characters for identification of species and the evidence for the separation of two sections of Cephalotaxus, namely: Cephalotaxus and Pectinatae. (3) The secondary phloem of this genus contains more or less crystalliferous parenchyma cells, in the inner tangential walls of which calcium oxalate crystals are embedded. So far this character has not been reported in the other families and the genera of conifers. Our conclusion, therefore, agrees with the opinion that Cephalotaxaceae is a natural taxon and includes only one genus, Cephalotaxus.     

Phloem Unloading within the Seed coat of Pisum sativum Observed using Surgically Modified Seeds

Phloem unloading in pea seed coats was observed by removingthe embryos from developing seeds and washing the attached coatswith a weakly buffered solution. The quantity of labelled photosynthateappearing in the washing solution varied immediately when thesolute concentration was changed, and is shown to be an osmoticresponse. This response is predicted by the Münch theoryof phloem transport with concentration dependent unloading.Respiratory inhibitors and the sulphydryl modifying reagentPCMBS had a slow effect upon the washout of tracer, which arrivedwithin the seed coat prior to inhibitor application, but completelystopped any washout of tracer arriving after its application.This time-course suggests that the inhibitors were not directlyinhibiting unloading, but preventing further tracer from enteringthe region of unloading within the seed coat. Phloem unloadingwithin the seed coats of Pisum appears to be passive and notdependent upon a PCMBS-sensitive carrier. Key words: Pisum sativum, seeds, phloem unloading     

Electron-Microscopic Evidence of the Vacuolar Nature of Phloem Exudate

We performed electron-microscopic examination of structural diurnal changes in the lumen of sieve tubes and the vacuolar system of corresponding companion cells and changes induced by the experimental blockage of assimilate export from the leaf by its cold-girdling. For these investigations, Cucurbita pepo L. and Helianthus annuus L. plants were used, that is, plant species from groups of symplastic and apoplastic plants, which differ in the type of companion cells and a mode of phloem terminal loading. The examinations showed the complete identity of changes in the electron texture of the sieve-tube lumens and companion-cell vacuoles in both plant species in the course of a day, when the level of assimilates changed, or after export blockage. Similar changes in the structure of the vacuolar labyrinths were stated in the companion cells under normal conditions and after cold-girdling, as related to the rate of sieve-tube loading with the vacuolar exudate. Vacuolar expansion and starch accumulation developing in response to changes in the assimilate level in the evening and after cold blockage of the assimilate export occurred in different types of cells, as dependent on their position in the symplast domains. However, the rate of the process similarly depended on the balance between assimilate synthesis and export. Synchronous changes in the texture of the sieve-tube lumen and companion-cell vacuoles were observed within each complex, but asynchronous changes occurred in different complexes. We suggested this phenomenon for recognizing the particular complexes, when they are grouped in a bundle. We observed no signs of cytoplasm or protein synthetic machinery in the sieve tubes. We concluded that the sieve-tube lumen and vacuoles of companion cells are common in nature. Similar electron texture of the images of the companion-cell vacuolar labyrinth and tube lumens, their connection through the lateral sieve fields, morphological modifications of the companion-cell vacuolar system as dependent on the activity of sieve tube loading—all of these facts imply the continuity of these transport compartments and fluxes in them and the similarity in the composition of the exudates from companion-cell vacuoles and phloem tubes.     

离心力对蚕豆叶片质外体汁液中磷酸己糖异构酶活力的影响

采用真空灌注离心法提取蚕豆叶片质外体汁液 ,通过测定不同离心力条件下叶片和叶片质外体汁液中磷酸己糖异构酶 (HPI)活力 ,判断蚕豆叶片质外体提取液是否受到细胞渗出物污染的结果表明 ,采用的适宜条件为 :离心温度≤ 4℃ ,离心力 5 0 0～ 80 0×g ,离心时间 8min。此条件下所获得的蚕豆叶片质外体汁液没有或很少受到细胞质的污染。