Sieve tube unloading and post-phloem transport of fluorescent tracers and proteins injected into sieve tubes via severed aphid stylets.

A variety of fluorescent tracers and proteins were injected via severed aphid stylets into the sieve tubes of wheat (Triticum aestivum L.) grains to evaluate the dimensions of plasmodesmal channels involved in sieve element/companion cell (SE/CC) unloading and post-phloem transport. In the post-phloem pathway, where diffusion is the predominant mode of transport, the largest molecule to show mobility was 16-kD dextran, with a Stokes radius of 2.6 nm. This suggests that the aqueous channels for cell-to-cell transport must be about 8 nm in diameter. Even the largest tracer injected into the sieve tubes, 400-kD fluorescein-labeled Ficoll with a Stokes radius of about 11 nm, was unloaded from the SE/CC complex. However, in contrast to smaller tracers (< or =3 kD, with a Stokes radius < or = 1.2 nm), the unloading of fluorescein-labeled Ficoll and other large molecules from the SE/CC complex showed an irregular, patchy distribution, with no further movement along the post-phloem pathway. Either the plasmodesmal channels involved in SE/CC unloading are exceptionally large (perhaps as much as 42 nm in diameter), with only a very small fraction of plasmodesmata being conductive, or the larger tracers damage the plasmodesmata in some way, enlarging smaller channels.

Gradients in water potential and turgor pressure along the translocation pathway during grain filling in normally watered and water-stressed wheat plants.

The water relations parameters involved in assimilate flow into developing wheat (Triticum aestivum L.) grains were measured at several points from the flag leaf to the endosperm cavity in normally watered (Psi approximately -0.3 MPa) and water-stressed plants (Psi approximately -2 MPa). These included direct measurement of sieve tube turgor and several independent approaches to the measurement or calculation of water potentials in the peduncle, grain pericarp, and endosperm cavity. Sieve tube turgor measurements, osmotic concentrations, and Psi measurements using dextran microdrops showed good internal consistency (i.e. Psi = Psi(s) + Psi(p)) from 0 to -4 MPa. In normally watered plants, crease pericarp Psi and sieve tube turgor were almost 1 MPa lower than in the peduncle. This suggests a high hydraulic resistance in the sieve tubes connecting the two. However, observations concerning exudation rates indicated a low resistance. In water-stressed plants, peduncle Psi and crease pericarp Psi were similar. In both treatments, there was a variable, approximately 1-MPa drop in turgor pressure between the grain sieve tubes and vascular parenchyma cells. There was little between-treatment difference in endosperm cavity sucrose or osmotic concentrations or in the crease pericarp sucrose pool size. Our results re-emphasize the importance of the sieve tube unloading step in the control of assimilate import.

S-methylmethionine plays a major role in phloem sulfur transport and is synthesized by a novel type of methyltransferase.

All flowering plants produce S-methylmethionine (SMM) from Met and have a separate mechanism to convert SMM back to Met. The functions of SMM and the reasons for its interconversion with Met are not known. In this study, by using the aphid stylet collection method together with mass spectral and radiolabeling analyses, we established that l-SMM is a major constituent of the phloem sap moving to wheat ears. The SMM level in the phloem ( approximately 2% of free amino acids) was 1.5-fold that of glutathione, indicating that SMM could contribute approximately half the sulfur needed for grain protein synthesis. Similarly, l-SMM was a prominently labeled product in phloem exudates obtained by EDTA treatment of detached leaves from plants of the Poaceae, Fabaceae, Asteraceae, Brassicaceae, and Cucurbitaceae that were given l-(35)S-Met. cDNA clones for the enzyme that catalyzes SMM synthesis (S-adenosylMet:Met S-methyltransferase; EC 2.1.1.12) were isolated from Wollastonia biflora, maize, and Arabidopsis. The deduced amino acid sequences revealed the expected methyltransferase domain ( approximately 300 residues at the N terminus), plus an 800-residue C-terminal region sharing significant similarity with aminotransferases and other pyridoxal 5'-phosphate-dependent enzymes. These results indicate that SMM has a previously unrecognized but often major role in sulfur transport in flowering plants and that evolution of SMM synthesis in this group involved a gene fusion event. The resulting bipartite enzyme is unlike any other known methyltransferase.

Variations of the natural abundances of nitrogen and carbon isotopes in Triticum aestivum, with special reference to phloem and xylem exudates.

This work explored whether the natural abundances of carbon and nitrogen isotopes could be used to describe the movement of C and X within wheat plants; we also considered whether isotopic analyses of aphids or their honeydew would substitute for direct analysis of phloem exudate. The δ13 C of ears and roots (sinks) most closely matched those of the sugars + organic acids fraction (sources) in both growth stages; phloem δ13 C matched that of leaf blade sugars. Xylem exudate δ13 C matched no other putative (and measured) source in the ear-forming stage and matched that of whole roots and ears in the grain-filling stage. The δ15 N of grain and roots (sinks) resembled that of leaf amino acids (sources) in the ear-forming stage. In the gram-filling stage, ear δ15 N continued to resemble that of leaf amino acids, and δ15 N of roots most closely resembled that of whole leaves. In the grain-filling stage, phloem δ15 N fell between that of leaf blade amino acids and that of whole leaves and was 15 X-depleted relative to internal and external NO, -N. In both growth stages, xylem exudate δ15 N was less than that of soil NO3 - -N and more than that of residual soil N after mineral N extraction. The isotopic values are generally in agreement with data from other approaches, such as isotope labelling; they show NO3 - -N reduction in both shoots and roots of wheat and significant N recycling (root-shoot-phloem-root) and C movement. Aphids might serve as a substitute for isotopic analysis of phloem δ15 N. having the same value as their food source. Their excreta was 15 N-enriched relative to phloem.

Post-phloem transport: principles and problems.

The movement of assimilates from the sieve element/companion cell complex to sites of utilization has been examined in an extensive array of sinks possessing diverse anatomies. This work has been reviewed with respect to the pathways taken, the conductances and driving forces for movement along the pathways, and interaction between the apoplast and symplast. Most investigations to date have been concerned primarily with determining the pathway of assimilate movement. A symplastic pathway is followed in the great majority of cases studied. However, available methods are less suited for demonstrating apoplastic transport in those instances where it occurs. Far less information is available on quantitative aspects of post-phloem transport. Only a very limited number of observations are available on the diffusive or hydraulic conductances of the apoplast or symplast. In some cases, symplastic conductance appears to be enhanced by a larger-than-usual size exclusion limit for cell-to-cell transport. Measurements of the driving forces for post-phloem transport (i.e. gradients in concentration and/or pressure) are also very few in number nor, to date, are they always readily interpretable. Evaluation of solute movement is complicated by interactions between the apoplastic and symplastic pathways, including water relations effects and solute exchange. The presence of apoplastic domains or, simply, high resistance to movement in the apoplast, can lead to steep water relations gradients within sinks, with important implications for transport. To understand how import into sinks is controlled, many more quantitative measurements are needed. This will require considerable experimental ingenuity.

Sucrose Release into the Endosperm Cavity of Wheat Grains Apparently Occurs by Facilitated Diffusion across the Nucellar Cell Membranes.

Nutrients required for the growth of the embryo and endosperm of developing wheat (Triticum aestivum L.) grains are released into the endosperm cavity from the maternal tissues across the nucellar cell plasma membranes. We followed the uptake and efflux of sugars into and out of the nucellus by slicing grains longitudinally through the endosperm cavity to expose the nucellar surface to experimental solutions. Sucrose uptake and efflux are passive processes. Neither was sensitive to metabolic inhibitors, pH, or potassium concentration. p-Chloromercuribenzene sulfonate, however, strongly inhibited both uptake and efflux, although not equally. Except for p-chloromercuribenzene sensitivity, these characteristics of efflux and the insensitivity of Suc movement to turgor pressure are similar to those of sucrose release from maize pedicels, but they contrast with legume seed coats. Although the evidence is incomplete, movement appears to be carrier mediated rather than channel mediated. In vitro rates of sucrose efflux were similar to or somewhat less than in vivo rates, suggesting that transport across the nucellar cell membranes could be a factor in the control of assimilate import into the grain.

Sucrose Concentration Gradients along the Post-Phloem Transport Pathway in the Maternal Tissues of Developing Wheat Grains.

Sucrose concentrations were measured in serial frozen sections of the post-phloem transport pathway in developing wheat (Triticum aestivum L.) grains. In normally importing grains, there was an approximately linear concentration gradient along the pathway, with a difference between the ends of the pathway of about 180 mM. This indicates an unusually low resistance for cell-to-cell transport, due perhaps to the large size-exclusion limit for the pathway. However, the existence of concentration gradients raises presently unresolvable questions about the relative contributions of diffusion versus bulk flow to transport within the symplast. The concentration gradient disappeared when sucrose movement ceased (i.e. in excised grains or when endosperm cavities of attached grains were perfused with p-chloromercuribenzene sulfonate [PCMBS] or with 1660 mOsm sorbitol). PCMBS appeared to block solute release into the endosperm cavity, whereas the sorbitol treatment, previously shown to cause localized plasmolysis in the chalaza, appeared to block movement across the chalaza. Sieve element/companion cell unloading appears to be an important control point for assimilate import. The sucrose concentration gradient and, probably, turgor and osmotic gradients are extremely steep there. PCMBS blocked import without affecting the sucrose concentration in the vascular parenchyma around the phloem. Thus, blockage of unloading was more complex than a simple "backing up" of solutes in the vascular parenchyma.

Health care reform based on an empowerment model of recovery by people with psychiatric disabilities.

People with psychiatric disabilities have articulated a model of recovery that encourages their empowerment by emphasizing consumer-defined goals, liberty, self-control of symptoms, peer support, elimination of discrimination, and provision of adequate material and social supports. Application of this model to health care reform requires public education to fight discrimination, an end to the use of involuntary interventions in the name of treatment, further development of services run by survivors-consumers and other alternatives to psychiatric hospitalization, and increased involvement of survivors-consumers in decisions related to their treatment and support. To promote empowerment of people with mental health problems, health care reform should include affordable, universal coverage without exclusions for preexisting high-risk conditions, parity of mental health benefits with other benefits, which includes coverage for voluntary services only, and incentives for funding long-term care, alternatives to hospitalization, and holistic healing services.

Monitoring Phloem Unloading and Post-Phloem Transport by Microperfusion of Attached Wheat Grains.

Phloem unloading and post-phloem transport in developing wheat (Triticum aestivum L.) grains were investigated by perfusing the endosperm cavities of attached grains. Relative unloading ratio (RUR) and the rate of sucrose release into the endosperm cavity (SRR) were calculated, respectively, from 14C import and from sucrose washout from the cavity. RUR and SRR continued at or near in vivo rates over a wide range of cavity sap osmolality (90 to approximately 500 milliosmolal) and sucrose concentration (14-430 mM) and for long times (29 h). These are much greater ranges than have been observed for the endosperm cavity in vivo (230-300 milliosmolal, and 40-120 mM, respectively), indicating that neither the cavity sap osmolality nor sucrose concentration are controlling factors for the rate of assimilate import into the cavity. The maintenance of in vivo transport rates over a wide range of conditions strongly implicates the role of transport processes within the maternal tissues of the wheat grain, rather than activities of the embryo or endosperm, in determining the rate of assimilate import into the grain. RUR was decreased by high concentrations of sucrose and sorbitol, but not of mannitol. By plasmolyzing some chalazal cells, sorbitol appeared to block symplastic transport across the crease tissues, but neither sucrose nor mannitol caused plasmolysis in maternal tissues of attached grains. The inhibition of RUR by KCN and carbonyl cyanide m-chlorophenyl (CCCP) and the continued import of sucrose into grains against its concentration gradient suggest that solute movement into the endosperm cavity might occur by active membrane transport. However, the evidence is weak, since KCN and CCCP appeared to act primarily on some aspect of symplastic (i.e. nonmembrane) transport. Also, sucrose could move from the endosperm cavity into the maternal tissues (i.e. opposite to the normal direction of sucrose movement), suggesting that transmembrane movement in the nucellus may be a reversible process. Pressure-driven flow into the grain could account for movement against a concentration gradient.

The Use of Fluorescent Tracers to Characterize the Post-Phloem Transport Pathway in Maternal Tissues of Developing Wheat Grains.

Various polar fluorescent tracers were used to characterize the pathways for apoplastic and symplastic transport in the "crease tissues" (i.e. the vascular strand, chalaza, nucellus, and adjacent pericarp) of developing wheat (Triticum aestivum L.) grains. With mostly minor exceptions, the results strongly support existing views of phloem unloading and post-phloem transport pathways in the crease. Apoplastic movement of Lucifer yellow CH (LYCH) from the endosperm cavity into the crease was virtually blocked in the chalazal cell walls before reaching the vascular tissue. However, LYCH could move slowly along the cell wall pathway from the chalaza into the vascular parenchyma. Slow uptake of LYCH into nucellar cell cytoplasm was observed, but no subsequent symplastic movement occurred. Carboxyfluorescein (CF) imported into attached grains moved symplastically from the phloem across the chalaza and into the nucellus, but was not released from the nucellus. In addition, CF moved in the opposite direction (nucellus to vascular parenchyma) in attached grains. Thus, the post-phloem symplastic pathway can accommodate bidirectional transport even when there is an intense net assimilate flux in one direction. When fresh sections of the crease were placed in fluorochrome solutions (e.g. LYCH or pyrene trisulfonate), dye was rapidly absorbed into intact cells, apparently via unsealed plasmodesmata. Uptake was not visibly reduced by cold or by respiratory inhibitors, but was greatly reduced by plasmolysis. Once absorbed, the dye moved intercellularly via the symplast. Based on this finding, a size-graded series of fluorescein-labeled dextrans was used to estimate the size-exclusion limits (SEL) for the post-phloem symplastic pathway. In most, and perhaps all, cells of the crease tissues except for the pericarp, the molecular diameter for the SEL was about 6.2 nm. The SEL in much of the vascular parenchyma may be smaller, but it is still at least 3.6 nm. Channel diameters would likely be about 1 nm larger, or about 4.5 to 7.0 nm in the vascular parenchyma and 7.0 nm elsewhere. These dimensions are substantially larger than those for "conventional" symplastic connections (about 3 nm), and would have a greater than proportionate effect on the per channel diffusive and hydraulic conductivities of the pathway. Thus, relatively small and probably ultrastructurally undetectable adjustments in plasmodesmatal structure may be sufficient to account for assimilate flux through the crease symplast.

A Kinetic and Microautoradiographic Analysis of [14C]Sucrose Import by Developing Wheat Grains.

Assimilates enter developing wheat grains via a strand of phloem extending along the crease region of the grain. After phloem unloading, they move several hundred micrometers before being released into the endosperm cavity, from which they are absorbed by the developing endosperm. Extraphloem assimilate pools in the maternal tissue of the crease, therefore, play a central role in post-phloem transport. We investigated the location and turnover of 14C-assimilates in the crease tissues and endosperm cavity sap by pulse labeling the flag leaf with 14CO2. Sucrose accounted for >90% of 14C at all times. Kinetic analysis of the crease sucrose pool and its depletion in excised grains showed that virtually the entire sucrose content of the crease tissues was involved in post-phloem transport and behaved basically as a single well-mixed compartment. Microautoradiographs also showed rapid movement of 14C throughout most of the crease tissues. Quantification of 14C concentration in the tissues showed a relatively shallow gradient of 14C and, presumably, of sucrose through the nucellus and chalaza. The steepest gradient in 14C content occurred in the vascular parenchyma between the chalaza and conducting cells (xylem and phloem).

Turnover of soluble proteins in the wheat sieve tube.

Although the enucleate conducting cells of the phloem are incapable of protein synthesis, phloem exudates characteristically contain low concentrations of soluble proteins. The role of these proteins and their movement into and out of the sieve tubes poses important questions for phloem physiology and for cell-to-cell protein movement via plasmodesmata. The occurrence of protein turnover in sieve tubes was investigated by [(35)S]methionine labeling and by the use of aphid stylets to sample the sieve tube contents at three points along a source-to-sink pathway (flag leaf to grains) in wheat plants (Triticum aestivum L.). Protein concentration and composition were similar at all sampling sites. The kinetics of (35)S-labeling of protein suggested a basically source-to-sink pattern of movement for many proteins. However, an appreciable amount of protein synthesis and, presumably, removal also occurred along the path. This movement appeared to be protein specific and not based on passive molecular sieving. The results have important implications for the transport capacities of plasmodesmata between sieve tubes and companion cells. The observations considerably expand the possible basis for ongoing sieve tube-companion cell interactions and, perhaps, interaction between sources and sinks.

Avoiding gas bubble formation during freeze substitution.

Gas bubbles frequently are formed during freeze substitution, especially when tissues are warmed to room temperature. The problem arises largely from the extreme solubility of CO2 in the freeze substitution solvent. Gas bubbles may be minimized by briefly transferring the tissue to freshly chilled solvent before warming to room temperature.

Measurement of Phloem transport rates by an indicator-dilution technique.

An indicator-dilution technique for the measurement of flow rates, commonly used by animal physiologists for circulation measurements, was adapted to the measurement of phloem translocation rates in the wheat (Triticum aestivum L.) peduncle. The approach is based on the observation that, during the transport of a given amount of solute, its mean concentration will be inversely proportional to flow rate. For phloem transport in the wheat peduncle, the necessary measurements are (a) the time course of tracer kinetics in the peduncle phloem, (b) the volume of sieve tubes and companion cells in the monitored segment of the peduncle, and (c) the amount of tracer transported past that point. The method was evaluated by in situ monitoring of (32)PO(4) transport in pulse-labeling experiments. Specific activities (i.e.(32)P concentrations) of phloem exudate were in good agreement with those calculated from in situ count rates and measured phloem areas. Mass transport rates, calculated from volume flow rates and phloem exudate dry matter content, also agreed well with expected mass transport rates based on measurements of grain growth rate and net CO(2) exchange by the ear. The indicator-dilution technique appears to offer good precision and accuracy for short-term measurements of phloem transport rates in the wheast peduncle and should be useful for other systems as well. In contrast to velocities based on time-delay measurements, it is more precise, more accurate, and produces an estimate of mean, rather than maximum, velocity. Also, since only a single detector is required, it can be applied to very short transport paths.

Accumulation and Conversion of Sugars by Developing Wheat Grains : VII. Effect of Changes in Sieve Tube and Endosperm Cavity Sap Concentrations on the Grain Filling Rate.

The extent to which wheat grain growth is dependent on transport pool solute concentration was investigated by the use of illumination and partial grain removal to vary solute concentrations in the sieve tube and endosperm cavity saps of the wheat ear (Triticum aestivum L.). Short-term grain growth rates were estimated indirectly from the product of phloem area, sieve tube sap concentration, and (32)P translocation velocity. On a per grain basis, calculated rates of mass transport through the peduncle were fairly constant over a substantial range in other transport parameters (i.e. velocity, concentration, phloem area, and grain number). The rates were about 40% higher than expected; this probably reflects some unavoidable bias on faster-moving tracer in the velocity estimates. Sieve tube sap concentration increased in all experiments (by 20 to 64%), with a concomitant decline in velocity (to as low as 8% of the initial value). Endosperm cavity sucrose concentration also increased in all experiments, but cavity sap osmolality and total amino acid concentration remained nearly constant. No evidence was found for an increase in the rate of mass transport per grain through the peduncle in response to the treatments. This apparent unresponsiveness of grain growth rate to increased cavity sap sucrose concentration conflicts with earlier in vitro endosperm studies showing that sucrose uptake increased with increasing external sucrose concentration up to 150 to 200 millimolar.

Amino Acid Composition Along the Transport Pathway during Grain Filling in Wheat.

The amino acid composition of endosperm cavity sap and of sieve tube saps from the flag leaf, peduncle, rachis, grain pedicel, and grain were determined for wheat plants just past the mid-half of grain filling. On a mole percent basis, glutamine accounted for almost half of the amino acids in sieve tube sap from the peduncle and ear. Other protein amino acids, plug gamma-aminobutyrate, were present in varying, but mostly low (a few mole percent) proportions. The amino acid composition of phloem exudate resembled that of the mature wheat grain. The proportions of amino acids in the endosperm cavity were generally similar to those of the sieve tube sap supplying the grain. Cysteine, however, while virtually absent from sieve tube sap, comprised 1 to 2 mole percent of amino acids in the endosperm cavity, suggesting it is transported in a different form. Also, alanine and, to a lesser extent, glutamate were relatively more prominent in endosperm cavity sap than in the sieve tube sap. Thus, while most amino acids were more concentrated in the sieve tube sap than in the endosperm cavity sap, alanine and glutamate appeared to be moving from the sieve tube to the endosperm cavity in the absence of, or perhaps even against, their concentration gradients.

Accumulation and Conversion of Sugars by Developing Wheat Grains : VI. Gradients Along the Transport Pathway from the Peduncle to the Endosperm Cavity during Grain Filling.

Gradients along the transport pathway from the peduncle to the endosperm cavity were examined during grain filling in wheat. Sieve tube exudate was collected from severed aphid stylets established on the peduncle and rachis and on the vascular bundles in the creases of grains. Phloem exudate could also be collected from broken grain pedicels, and by puncturing the vascular bundle in the grain crease with a needle. Stylets on excised grains persisted exuding, indicating that grain sieve tubes are capable of loading solutes. There was little, if any, discernible gradient in osmolality or solute composition (sucrose, total amino acids) of sieve tube contents along the phloem pathway from the peduncle to the rachis or along the rachis itself. Neither was a gradient detected in osmolality along the sieve tube pathway from the rachis through the rachilla and grain stalk to the crease. Demonstrable solute gradients occurred only across those tissues of the grain crease between the crease sieve tubes and the endosperm cavity, a distance of just 1 millimeter. However, while the sucrose concentration in the sieve tubes was almost tenfold that in the endosperm cavity sap, total amino acids were only threefold higher, and the potassium concentrations of the two were equal. Our observations strongly implicate the movement of assimilates from the sieve tubes and across the crease tissues as important control points in grain filling.

In situ measurement of plant water potentials by equilibration with microdroplets of polyethylene glycol 8000.

Microdroplets (3-5 nanoliters) of polyethylene glycol 8000 solution were allowed to equilibrate with plant water potential by placing the microdroplet on an abraded surface and covering it with mineral oil to prevent evaporation. Osmolality was followed by cryoscopic measurements, accurate to about +/-0.1 bar, on subnanoliter samples.Under constant environmental conditions, apparent equilibrium between microdroplet and plant water potentials was attained in about 1 to 2 hours. Microdroplet osmolality responded promptly to treatments (illumination, excision, osmotica) which changed plant water status. The values obtained for plant water potentials appeared to be physiologically reasonable. However, comparison with values obtained by other means (dewpoint hygrometry, treatment of tissue with polyethylene glycol solutions, calculation from turgor and osmotic pressures) suggest that they might be somewhat more negative than the actual tissue water potential.Aside from the advantage of providing in situ measurements of plant water status, the method is not temperature sensitive and requires only about 10 square millimeters of surface area, which allows its use on even small structures with little interference by shading or with gas exchange.