Donald B. Fisher

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 (∼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–35S-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 (∼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.

A guide to the use of the exuding-stylet technique in phloem physiology

The use of exuding stylets holds considerable promise for the investigation of sieve-tube physiology. However, largely because of difficulties in cutting insect stylets, the technique has been applied to only a few plant species. Based on our experience, a comparison is made of the available means of obtaining sieve-tube exudate from the exuding stylets of phloem-feeding insects, including aphids, scale and mealybugs. Forty-one plant species and approx. 35 insect species were tested for their ability to provide stylet exudate. Stylets on all but a few of the plant species tested yielded at least some exudate, but the success rate and duration of exudation on many species were unsatisfactory for detailed investigations of phloem transport. Plant species appears to be the most important factor for obtaining reliably exuding stylets, although the size of the insect species used and the physiological condition of the plant are also important variables. Armored scale provide a simple and reliable source of exuding stylets, but are impractical for most experimental purposes. Radio-frequency microcautery of aphid stylets was substantially the most effective means of cutting stylets. Instructions are provided for constructing a microcautery unit at minimal expense, using a citizens band radio as the radio-frequency source.

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.

Osmoregulation by the aphid Myzus persicae: A physiological role for honeydew oligosaccharides

The dry matter content of honeydew produced by Myzus persicae feeding on artificial diets increased with increasing sucrose concentrations of the diet. Whereas the diet osmolalities ranged from 828 to 1800 milliosmolal, the honeydew osmolality was relatively constant (about 500 milliosmolal). This osmoregulatory capacity is achieved largely by variations in the mean molecular weight of glucose-containing honeydew oligosaccharides.

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.

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.

Measurement of aphid feeding rates on artificial diets using 3H‐inulin

The radioactivity of the honeydew droplets excreted by young apterous adults of the green peach aphid, Myzus persicae, fed on an artificial diet containing 3H‐inulin was a reliable measure of the volume of food ingested by the insects, since almost none of the ingested inulin was absorbed and retained by the insects.

Year-round collection of willow sieve-tube exudate

Seasonal variations in sieve-tube sap were followed by collecting exudate from the broken stylets of an armored scale insect (Quadraspidiotus ostreaformis Curtis) which overwintered on 3- to 6-year-old branches of a local willow population (Salix exigua Nutt.). During the winter, the scale insects fed on functional sieve tubes near the cambium. Broken stylets on stem segments taken to the laboratory exuded promptly even when collections were made at temperatures as low as-30°C. Exudation in the field was observed at-11 to-13°C. Sucrose accounted for virtually all of the sugars in exudate collected during the winter. Sieve-tube sap collected during the winter was about twice as concentrated as summer sievetube sap. The concentration decreased during rapid vegetative growth in the spring, and increased again as growth slowed in late summer and early fall.

The estimated pore diameter for plasmodesmal channels in the Abutilon nectary trichome should be about 4 nm, rather than 3 nm

Abstract. By injecting varous-size fluorescent probes into Abutilon striatum Dicks nectary trichomes, Terry and Robards (1987, Planta 171: 145–157) arrived at an estimate of 3 nm for the physical diameter of the channels involved in cell-to-cell movement. However, their calculations contain an arithmetic error in the volume assumed for the cell into which the probes diffused. (The actual volume is ten times larger.) The corrected calculations give a physical diameter of about 4 nm.

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).