Effect of Water Stress on Turgor Differences and C-Assimilate Movement in Phloem of Ecballium elaterium

The effects of water stress on pressure differences and ¹⁴C-assimilate translocation in sieve tubes of squirting cucumber Ecballium elaterium A. Rich were studied. Water stress was induced by transfer of plants from culture solution to a polyethylene glycol 6,000 solution having an osmotic potential of −18.2 atm. Sieve tube turgor, turgor differences between source and sink, and translocation rate were decreased. After 260 minutes of translocation, only 19% of the total fixed ¹⁴CO₂ had moved out of the leaf, compared to the control value of 62% after the same period of time. The results suggest that water stress slows translocation by lowering sieve tube turgor differences, which are essential for the pressure flow mechanism of conduction.     

Turgor-dependent unloading of assimilates from coats of developing legume seed. Assessment of the significance of the phenomenon in the whole plant

The in vivo significance of turgor-dependent unloading was evaluated by examining assimilate transport to and within intact developing seeds of Phaseolus vulgaris (cv. Redland Pioneer) and Vicia faba (cv. Coles Prolific). The osmotic potentials of the seed apoplast were low. As a result, the osmotic gradients to the seed coat symplast were relatively small (i.e. 0.1 to 0.3 MPa). Sap concentrations of sucrose and potassium in the seed apoplast and coat symplast accounted for some 45 to 60% of the osmotic potentials of these compartments. Estimated turnover times of potassium and sucrose in the seed apoplast of < 1 h were some 5 to 13 times faster than the respective turnover times in the coat symplast pools. The small osmotic gradient between the seed apoplast and coat symplast combined with the relatively rapid turnover of solutes in the apoplast pool, confers the potential for a small change in assimilate uptake by the cotyledons to be rapidly translated into an amplified shift in the cell turgor of the seed coat. Observed adjustments in the osmotic potentials of solutions infused between the coat and cotyledons of intact seed were consistent with the in vivo operation of turgor-dependent unloading of solutes from the coat. Homeostatic regulation of turgor-dependent unloading was indicated by the maintenance of apoplast osmotic potentials of intact seeds when assimilate balance was manipulated by partial defoliation or elevating pod temperature. In contrast, osmotic potentials of the coat symplast adjusted upward to new steady values over a 2 to 4 h period. The resultant downward shift in coat cell turgor could serve to integrate phloem import into the seed coat with the new rates of efflux to the seed apoplast. Circumstantial evidence for this linkage was suggested by the approximate coincidence of the turgor changes with those in stem levels of ³²P used to monitor phloem transport. The results obtained provide qualified support for the in vivo operation of a turgor homeostat mechanism. It is proposed that the homeostat functions to integrate assimilate demand by the cotyledons with efflux from and phloem import into the coats of developing legume seed.     

Loading and translocation of assimilate in the fine veins of sunflower leaves

Abstract The time course of loading and transport of assimilate in sunflower leaves was examined by pulse labelling with ¹⁴CO₂, followed by freeze drying or freeze substitution, and dry autoradiography at both low and high resolution. The five classes of veins, V1-V5 (V5 being smallest), show a division of function: V5 and V4 are engaged in loading and short distance transport; V3 to V1, in long distance translocation. The first high concentration of ¹⁴C is found in two or three phloem parenchyma cells (intermediary cells) of V5 and V4 veins. The sieve elements of V5 and V4 veins do not show comparable concentrations of ¹⁴C at any time. Recently assimilated ¹⁴C is transported by the intermediary cells for distances of about 0.5 mm to the V3 veins. In V3 to V1 veins translocation is in the sieve tubes. Transport in V5 and V4 veins is in two directions, that in V3 to V1, in one direction towards the petiole. The high concentration of ¹⁴C formed in the intermediary cells does not increase further as the assimilate moves to the sieve tubes of the V3 veins, and so is probably the origin of the gradient that drives translocation.     

The effect of irradiance, p-chloromercuribenzenesulphonic acid and fusicoccin on the long distance transport in Zea mays L. seedlings

The system consisting of a few proportional detectors with appropriate electronic components was earlier developed for in vivo studies of long distance transport in whole maize seedlings. ¹⁴CO₂ assimilation rate (P_a), time of radioactivity appearing in the loading region (AT), transport speed in the leaf (TS_l), transport speed between the leaf and the roots (TS_r), the maximum radioactivity values detected in the leaf below the feeding area (R_l) and in the mesocotyl (R_r) from leaves to roots in maize seedlings were calculated from the obtained temporal profiles of radioactivity. The study was undertaken to follow the changes in separate steps of long distance transport in maize seedlings as affected by two light irradiances and application of p-chloromercuribenzenesulphonic acid and fusicoccin, with the aim to investigate different steps of long distance transport, particularly phloem loading. The method used allows to study in vivo the different aspects of long distance transport in maize seedlings, both qualitatively and quantitatively. It was shown that the characteristics obtained from the radioactivity profiles corresponded to different steps of long distance transport, as assimilate synthesis, phloem loading, and phloem translocation. It was also demonstrated that although active phloem loading participate in assimilate export from the leaves, assimilate transport along the maize seedling might undergo accordingly to assimilate gradient, particularly under light irradiance higher than during the growth.     

Measurement of turgor pressure and its gradient in the Phloem of oak

A direct method is described for measuring the pressure in secondary phloem sieve tubes of oak trees. One end of a 26-gauge stainless steel tube was shaped such that when it penetrated the outer bark and transected a few sieve elements, it was stopped by the xylem so that small openings in the end allowed phloem sap to enter the tube. The other end of the stainless tube (phloem needle) was joined to a long glass capillary sealed at its other end to form a manometer for measuring phloem sap pressure. A method for measuring the average osmotic and turgor pressures in cells of leaves is also described. Phloem turgor pressures varied greatly in a series of phloem punctures around the trunk at 1.5 and at 6.3 meters. The variation in turgor pressure was always greater than the variation in osmotic pressure. In a series of turgor pressures arranged in descending order, the values in a sequence for the upper level was usually a little (0-3 atm) larger than the values for the lower level. These results may suggest that translocation of assimilate is favored by a small turgor pressure gradient, but they do more to emphasize the complications in measuring gradients in an elastic low resistance distribution system composed of contiguous longitudinal conduits. The results also imply that the sieve tubes are inflated with assimilate fluid under high pressure which can readily move longitudinally and with less pressure drop than would be necessary if the sieve tubes were rigid.     

Effect of nitrate infusion into the shoot apoplast on photosynthesis and assimilate transport in symplastic and apoplastic plants

The shoots of fireweed (Chamerion angustifolium (L.) Holub) and common flax (Linum usitatissimum L.) were infused with 50 mM KNO₃ solution to compare the influence of nitrate on photosynthesis and assimilate export from leaves in plants with the symplastic and apoplastic phloem loading, respectively. The infusion of nitrate in the shoots of both plant species lowered ¹⁴CO₂ fixation and enhanced the assimilate transport in the upward direction. Irrespective of the phloem loading type, the incorporation of ¹⁴C into sucrose decreased in nitrate-treated seedlings exposed to assimilation for short (3 min) periods. However, when shoots were sampled 3 h after ¹⁴CO₂ fixation, the content of ¹⁴C-labeled sucrose was higher in treated plants than in control seedlings infused with water. In fireweed, in contrast to flax, a similar temporal pattern was also characteristic for ¹⁴C incorporation into oligosaccharides. Within 3 h after nitrate infusion into the fireweed apoplast, the mitochondria and the cell vacuolar system underwent ultrastructural changes indicative of the increase in cytosolic osmotic pressure. At the same time, we observed accumulation of fibrillar inclusions in cell vacuoles of vascular bundles. It is concluded that the mechanisms of nitrate influence on photosynthesis and sugar export in leaves of symplastic and apoplastic plants are similar to a certain extent and involve the blocking of pores in phloem tubes, initiated by the NO-signaling system.     

Interaction of cell turgor and hormones on sucrose uptake in isolated Phloem of celery

Phloem tissue isolated from celery (Apium graveolens L.) was used to investigate the regulation of sucrose uptake by turgor (manipulated by 50-400 milliosomolal solutions of polyethylene glycol) and hormones indoleacetic acid (IAA) and gibberillic acid (GA₃). Sucrose uptake was enhanced under low cellular turgor (increase in the V_max). Furthermore, enhancement of sucrose uptake was due to a net increase in influx rates since sucrose efflux was not affected by cell turgor. Manipulations of cell turgor had no effect on 3-O-methyl glucose uptake. When 20 millimolar buffer was present in uptake solutions, low turgor-induced effects were observed only at low pH range (4.5-5.5). However, the effect was extended to higher external pH (up to 7.5) when buffer was omitted from uptake solutions. A novel interaction between cellular turgor and hormone treatments was observed, in that GA₃ (10 micromolar) and IAA (0.1-100 micromolar) enhanced sucrose uptake only at moderate turgor levels. The hormones elicited little or no response on sucrose uptake under conditions of low or high cell turgor. Low cell turgor, IAA, GA₃, and fusicoccin caused acidification by isolated phloem segments in a buffer-free solution. It is suggested that enhanced sucrose uptake in response to low turgor and/or hormones was mediated through the plasmalemma H⁺-ATPase and most likely occurred at the site of loading.     

Sink to source translocation in soybean

The possibility that phloem loading may occur in the reproductive sink tissues of soybeans (Glycine max Merr. cv Chippewa 64) was examined. When [¹⁴C]sucrose was applied to seed coat tissues from which the developing embryo had been surgically removed, 0.1% to 0.5% of the radioactivity was translocated to the vegetative plant parts. This sink to source translocation was largely unaffected by destroying a band of phloem with steam treatment on the stem above and below the labeled pod. The same steam treatment, however, completely abolished translocation of [¹⁴C]sucrose between mature leaves and developing fruits. These results indicate that the movement of nutrients from developing seed coats to the vegetative plant parts occur in the xylem and that phloem loading does not occur in this sink tissue.     

Potassium in the Apoplast of the Root Sink Region

Using carboxyfluorescein, a fluorochrome transported along the phloem, we demonstrated that symplasmic phloem unloading in the watermelon root occurred in the basal zone of the meristem adjusting to the elongation zone. In the similar zones of maize and pumpkin roots, a high level of potassium was detected by X-ray microanalysis in the cell walls and intercellular spaces. Potassium concentration in these compartments comprised two-thirds of that in the cytoplasm. Such proportion between potassium concentrations in the cytoplasm and apoplast was characteristic of both the cortex and stele. Since potassium is a dominant osmotically active component in root tissues, such a proportion between its intracellular and apoplastic concentrations provides for a low turgor pressure in the cells of the sink region, in the phloem in particular. This might increase a turgor pressure gradient along the translocation route between source and sink tissues, which is a driving force for phloem assimilate transport.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 591–599.Original Russian Text Copyright © 2005 by Krasavina, Burmistrova, Feshchenko, Nosov.     

Spontaneous Phloem bleeding from cryopunctured fruits of a ureide-producing legume

The vasculature of the dorsal suture of cowpea (Vigna unguiculata [L.] Walp) fruits bled a sugar-rich exudate when punctured with a fine needle previously cooled in liquid N₂. Bleeding continued for many days at rates equivalent to 10% of the estimated current sugar intake of the fruit. A phloem origin for the exudate was suggested from its high levels (0.4-0.8 millimoles per milliliter) of sugar (98% of this as sucrose) and its high K⁺ content and high ratio of Mg²⁺ to Ca²⁺. Fruit cryopuncture sap became labeled with ¹⁴C following feeding of [¹⁴C]urea to leaves or adjacent walls of the fruit, of ¹⁴CO₂ to the pod gas space, and of [¹⁴C] asparagine or [¹⁴C]allantoin to leaflets or cut shoots through the xylem. Rates of translocation of ¹⁴C-assimilates from a fed leaf to the puncture site on a subtended fruit were 21 to 38 centimeters per hour. Analysis of ¹⁴C distribution in phloem sap suggested that [¹⁴C]allantoin was metabolized to a greater extent in its passage to the fruit than was [¹⁴C] asparagine. Amino acid:ureide:nitrate ratios (nitrogen weight basis) of NO₃-fed, non-nodulated plants were 20:2:78 in root bleeding xylem sap versus 90:10:0.1 for fruit phloem sap, suggesting that the shoot utilized NO₃-nitrogen to synthesize amino acids prior to phloem transfer of nitrogen to the fruit. Feeding of ¹⁵NO₃ to roots substantiated this conclusion. The amino acid:ureide ratio (nitrogen weight basis) of root xylem sap of symbiotic plants was 23:77 versus 89:11 for corresponding fruit phloem sap indicating intense metabolic transfer of ureide-nitrogen to amino acids by vegetative parts of the plant.     

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.     

Translocation of sugar and tritiated water in squash plants

When ¹⁴C-sugar and THO were simultaneously introduced through a cut side vein or flap of a squash leaf (Cucurbita melopepo, Bailey cv. torticollis) concurrent translocation of ¹⁴C-sugars, T-photosynthates and THO with parallel, almost flat, gradients was observed in the petiole for periods of 1 to 3 hr. Parallel translocation gradients were not observed when ¹⁴C was introduced as ¹⁴CO₂ and T by painting a leaf with THO. Autoradiography of frozen sections to locate the tissues in which THO was moving was unsuccessful. Steam-girdling blocked the movement of ¹⁴C and T when ¹⁴C-glucose and THO were introduced simultaneously by the flap-feeding technique. If THO moved as liquid water in the phloem along with the ¹⁴C-sugars, as blockage by steam girdling suggests, then solution flow of sugar cannot be excluded as a mechanism of translocation.     

Osmoregulation in Cotton Fiber: Accumulation of Potassium and Malate during Growth

Kinetics and osmoregulation of cotton (Gossypium hirsutum L.) fiber growth (primarily extension) have been studied. Growth is dependent on turgor pressure in the fiber. It is inhibited when a decrease in the water potential of the culture medium due to an addition of Carbowax 6000, equals the turgor pressure of the fiber. Potassium and malate accumulate in the fiber and reach peak levels when the growth rate is highest. Maximum concentrations of potassium and malate reached in the fiber can account for over 50% of the osmotic potential of the fiber. As growth slows down, levels of potassium and malate decrease and turgor pressure declines. Cotton ovules are capable of fixing H¹⁴CO₃⁻ in the dark, predominantly into malate. Fiber growth is inhibited by the absence of potassium and/or atmospheric CO₂. We suggest that potassium and malate act as osmoregulatory solutes and that malate, at least in part, arises from dark CO₂ fixation reactions.     

The Response of Groundnut (Arachis hypogaea L.) to Timing of Irrigation: II. 14C-PARTITIONING AND PLANT WATER STATUS

Finite quantities of water were applied at different growthstages of groundnut stands (Arachis hypogaea L.) grown in controlledenvironment glasshouses. Soil moisture deficits were imposedbetween sowing and pod initiation or between pod initiationand final harvest by withholding or applying water. Effectson assimilate production and partitioning and plant water relationswere examined. Leaves were the primary sites of ¹⁴CO₂ fixation, though theircontribution generally declined late in the season, whereasfixation by stems was initially low but increased sharply whenstress was released in the late-irrigated stands. ¹⁴C-fixationby stem apices and pegs also rose sharply following irrigationof the late-stressed stands. Leaves were the primary source of assimilates, though translocationtended to decrease as the season progressed, even in the late-irrigatedstands. Stems were initially the major sinks, but their sinkactivity disappeared almost completely when stress was releasedin the late-irrigated stands. Assimilate import by stem apicesdeclined progressively and pod sink activity was negligiblein the late-stressed stand, but both increased markedly whenearly-season stress was released. Leaf water status showed marked diurnal variation, whereas pegsshowed less variation and maintained much higher turgor levels,largely because of their lower solute potentials. Marked osmoticadjustment occurred in expanding but not in mature leaves, allowingthem to maintain higher turgor levels during periods of severestress. This adjustment was rapidly lost when stress was released.The observed changes in assimilate production and partitioningpreceded detectable changes in bulk turgor levels. Implications for growth, development and yield are discussed. Key words: Groundnut, irrigation, partitioning, water status     

Translocation of Carbohydrate in Cotton: Movement to the Fruiting Bodies

The translocation of ¹⁴C-labelled assimilate was followed frommain stem leaves on the cotton plant (Gossypium hirsutum) tobolls on the lower sympodia. From the upper leaves the assimilatedescends the stem in well-defined strands in the phloem. Wheresympodia originate close to these strands some of the assimilateis distributed to the bolls on those sympodia. There is evidencethat the involucre, but not the green boll wall, contributesto boll nutrition by assimilation of external CO₂.     

Evidence for Phloem loading from the apoplast: chemical modification of membrane sulfhydryl groups

The water-soluble, sulfhydryl-specific, chemical modifier p-chloromercuribenzenesulfonic acid reversibly inhibited the accumulation of exogenously supplied ¹⁴C-sucrose into leaf discs of Beta vulgaris. P-Chloromercuribenzenesulfonic acid treatment did not inhibit photosynthesis or respiration or induce membrane leakage to sucrose, indicating that the site of inhibition was the plasmalemma. The active loading of sucrose and ¹⁴CO₂-derived assimilates into the phloem and their translocation from the source leaf were inhibited by the nonpermeant modifier. Several nonpermeant sulfhydryl group modifiers also inhibited sucrose accumulation into leaf discs while two amino-reactive reagents had no effect. The results indicate that sugars are actively accumulated into the phloem from the apoplast and that membrane sulfhydryl groups may be involved.     

The effect of cell turgor on sugar uptake in strawberry fruit cortex tissue

A reduction in cell turgor has been shown to stimulate sugar uptake in several plant sink tissues and it may regulate the import of assimilate into the sink apoplast, as well as maintain cell turgor. To determine whether cell turgor influences sugar uptake by strawberry (Fragaria x ananassa Duch. cv. Brighton) fruit cortex tissue, disks were cut from greenhouse-grown primary fruit at the green-white stage of development and placed in buffered incubation solutions containing either mannitol or ethylene glycol as an osmoticum. Cell turgor of fruit disks was calculated from the difference between the water potential of bathing solution and tissue solute potential after incubation at various osmolarities. Cell turgor increased when tissue disks were placed into mannitol incubation solutions more dilute than the water potential of fresh tissue (about 415 mOsmol kg^?1). The rate of uptake of [¹⁴C]-sucrose or [¹⁴C]-glucose decreased as osmolarity of the incubation solution increased, i.e. as cell turgor declined. Cell turgor and the rate of [¹⁴C]-sucrose uptake were unaffected when rapidly permeating ethylene glycol was used as an osmoticum. A decrease in cell turgor reduced both the V_max of the saturable (carrier mediated) kinetic component of sucrose uptake, and the slope of the linear (diffusional) component. The sulfhydryl binding reagent p-chloromercuibenzenesulfonic acid, an inhibitor of the plasma membrane sucrose carrier, strongly inhibited only the saturable component of sucrose uptake. Increased uptake of the nonmetabolizable sugar, O-methyl-glucose, at high turgor was similar to that of glucose, indicating that carrier activity was influenced by cell turgor, not cell metabolism. Turgor did not influence efflux of [¹⁴C]-sucrose from disks and had no effect on cell viability. Strawberry fruit cells do not possess a sugar uptake system that is stimulated by a reduction in turgor.     

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.     

Translocation and accumulation of translocate in the sugar beet petiole

Accumulation of translocate during steady-state labeling of photosynthate was measured in the source leaf petioles of sugar beet (Beta vulgaris L. monogerm hybrid). During an 8-hr period, 2.7% of the translocate or 0.38 μg carbon/min was accumulated per cm petiole. Material was stored mainly as sucrose and as compounds insoluble in 80% ethanol. The minimum peak velocity of translocation approached an average of 54 cm/hr as the specific activity of the ¹⁴CO₂ pulse was progressively increased. The ratio of cross sectional area required for translocation to actual sieve tube area in the petiole was 1.2. A regression analysis of translocation rate versus sieve tube cross sectional area yielded a coefficient of 0.76. The specific mass transfer rate in the petiole was 1.4 g/hr cm² phloem or 4.8 g/hr cm² sieve tube. Histoautoradiographic studies indicated that translocation occurs through the area of phloem occupied by sieve tubes and companion cells while storage occurs in these cells plus cambium and phloem parenchyma cells. The ability of the petiole to act as a sink for translocate is consistent with the concept that storage along path tissue serves to buffer sucrose concentration in the translocate during periods of fluctuating assimilation.     

Transport processes in stimulated and non-stimulated leaves ofMimosa pudica

Summary Mature leaves ofMimosa pudica L. or parts of them were exposed to¹⁴CO₂, and translocation was recorded by macroautoradiography. It was observed that considerable amounts of labelled photoassimilates were accumulated in pulvini when the leaf was stimulated. In non-stimulated leaves, no such accumulation of label was observed.Microautoradiographs of pulvinar regions of the non-stimulated leaf showed¹⁴C- label restricted to the phloem. When stimulated, the¹⁴C- label was unloaded from the phloem of the pulvini. Labelled photoassimilates appeared most concentrated in the walls of the collenchymatous cells and beyond in the extensor region of the motor cortex. There, label was accumulated in the apoplastic compartments. Stimulation causes a sudden phloem unloading of sucrose, and its accumulation in the apoplast lowers the water potential which eventually exceeds the osmotic potential of the extensor cells of the motor cortex. By removal of cytoplasmic water the motor cells lose turgidity which results in the closing movement of the leaflets, and — some seconds later — in the bending down of the petiole. In late afternoon night-stimulation triggers sucrose unloading in secondary pulvini. During phases of relaxation, labelled material is taken up by motor cells of the extensor, which concomitantly gain turgor.Part of the doctoral dissertation of Jörg Fromm supported by the Deutsche Forschungsgemeinschaft