Analysing partitioning of recently fixed and of reserve carbon in reproductive Phaseolus vulgaris L. plants

Phloem import and unloading in perfused bean (Phaseolus vulgaris L.) seed coats were investigated using steady-state labeling. Though photosynthate import and unloading were significantly reduced by perfusion, measurements of photosynthate fluxes in perfused seed coats proved useful for the study of unloading mechanisms in vivo. Phloem import was stimulated by lowered seed coat cell turgor, as demonstrated by an increase in tracer and sucrose import to seed coats perfused with high concentrations of an osmoticum. The partitioning of photosynthates between retention in the seed coat and release to the perfusion solution also was turgor sensitive; increases in seed coat cell turgor stimulated photosynthate release to the apoplast at the expense of photosynthate retention within the seed coat. There was no evidence of a turgor-sensitive sucrose uptake mechanism in perfused seed coats. Thus, the turgor sensitivity of photosynthate partitioning within perfused seed coats was consistent with a turgor-sensitive efflux control mechanism. Measurements of tracer equilibration and sugar partitioning in perfused seed coats provided strong evidence for symplastic phloem unloading in seed coats.The empty seed coat technique permits the analysis of photosynthates released from maternal seed tissues following the surgical removal of the embryo and the addition of a trapping solution or agar (12,18,22). Sucrose is quantitatively the most important material eluted from perfused seed coats of species studied to date (12,17,18,21,22).The efflux of pulse-labeled photosynthates and unlabeled sugars (and amino acids) from seed coats attached to the plant via the funiculus is enhanced by perfusion with solutions containing high concentrations of an osmoticum (1,6,10,(23)(24)(25)(26). The mass flow hypothesis of phloem transport predicts that lowering the turgor pressure ofsieve elements within sink tissues will enhance phloem import into sinks (7). Osmoticum concentration does not influence seed coat membrane permeability (determined by ['4C]mannitol-uptake ex-

Section: Effects Of Perfusion On Photosynthate Import and Efflux Fromcontrasting

confidence: 53%

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L.

Ellis¹,

Turgeon²,

Spanswick³

1992

“…To measure accumulation of tracer carbon, four 0.13-cm2 leaf samples were taken from leaf 2 at hourly intervals throughout the light period (13). Samples were removed through a small opening in the leaf chamber lid to lessen the entry of unlabeled CO2 and the consequent lowering of SR. After the samples were dried, weighed and oxidized in scintillation vials (1,10), their 14C content was assayed by liquid scintillation counting and the resulting data, along with measured SR, were used to calculate leaf tracer carbon. Tracer carbon content of the leaf being monitored by the G-M detector also was determined from several samples taken at the end of the day.…”

Section: Methodsmentioning

confidence: 99%

A Method for Calculating Sucrose Synthesis Rates throughout a Light Period in Sugar Beet Leaves

Geiger¹,

Fondy²,

Tucci³

1988

Sucrose synthesis rate in an exporting sugar beet (Beta vulgaris L.) leaf was calculated from simultaneous measurements of export and changes in leaf sucrose level. The amount of recently fixed carbon exported was determined from net carbon assimilated minus the tracer carbon accumulated in the leaf. The relative amount of "4C accumulated in the leaf supplied with '4CO2 throughout an entire light period was recorded continuously with a Geiger-Mueller detector. To produce a continuous time course for tracer carbon accumulated in the leaf during the light period, the latter curve was superimposed on values for tracer carbon accumulated in leaves sampled at hourly intervals. Validity of the method requires that nearly all of the carbon that is exported be sucrose and that nearly all of the sucrose that is synthesized be either exported or accumulated as sucrose in the exporting leaves. These conditions appeared to be fulfilled in the situations where the method was applied. The method was used to study the effect of increasing atmospheric CO2 concentration on the rate of sucrose synthesis. Further, the method can be used in conjunction with the gathering of other data such as gas exchange, metabolite levels, and enzyme activities in a set of leaves of a similar age on the same plant. This assemblage of data was found to be useful for understanding how rates of photosynthesis, sucrose synthesis, and translocation are regulated in relation to each other in an intact plant.on steady state labeling (7,8,12,13), for calculating rates of sucrose synthesis throughout a daily light period in leaves of sugar beet, a sucrose-exporting plant. Measurements required for estimating sucrose synthesis were designed to be done in conjunction with concurrent measurements of gas exchange, metabolite levels, and enzyme activities in a set of similar leaves on the same plant. Changes in sucrose synthesis produced by experimental treatment were measured by the method.MATERIALS AND METHODS Plant Material. Sugar beet plants (Beta vulgaris L., Klein E type, multigerm) were grown in 6-L containers in a mixture of Jiffy Mix (Ball Jiffy Co.):sand (1: 1, v/v) watered three times daily with a nutrient solution (4). Plants were maintained under a 14-h photoperiod at 25°C, 60% RH day and 17°C, 75% RH night. Typically, leaves of plastochrons 4 and 5, with an area of 1.0 to 1.5 dm2, were used when plants were 4 weeks old.Irradiance. Maximum irradiance was 0.75 mmol m-2 S' at leaf blade level during growth and during experiments. During growth, light intensity was changed in 3 ascending and 3 descending steps to approximate natural diurnal irradiance. During experiments a 12 x 0.6 m mylar strip, consisting of a closely graded series of neutral density filters, was moved between the light source and the plant to produce a sinusoidal light regime. The target irradiance level provided by computer-directed movement of the filter strip is given by the equation: Sucrose synthesis has been the subject of much research interest recently because of i...

“…Steady-state labeling has been used for the quantitative analysis of various aspects of carbon partitioning in plants because the stable tracer fluxes produced by the continuous assimilation of "'CO2 at constant specific activity result in relatively simple tracer kinetics at the sink (7,9).…”

mentioning

confidence: 99%

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L.

Ellis

Turgeon²,

Spanswick³

1992

The pathway and kinetics of photosynthate unloading in developing seeds of bean (Phaseolus vulgaris L.) were investigated using steady-state labeling with '4C02. The continuous assimilation of 14CO2 at constant specific activity produced stable tracer fluxes that facilitated straighiforward analyses of photosynthate import and unloading in developing seeds. The kinetics of tracer equilibration within intact seeds were compatible with a symplastic route of photosynthate unloading in the seed coat. The import and partitioning of tracer within seeds were partially disrupted by the surgical excision of the distal halves of seeds as practiced during the preparation of "empty" seed coats for perfusion.The mechanisms that regulate photosynthate allocation within developing legume seeds have been the focus of many recent studies (for reviews see refs. 16, 25, 30). Photosynthates accumulated by the embryo must first be unloaded to the seed coat apoplast because the seed coat and embryo of legume seeds are not symplastically linked (25). The pathway of unloading to the seed coat apoplast has not been conclusively determined. Theoretically, photosynthates could unload directly from the phloem to the apoplast (apoplastic pathway) (29, 30), or they could move symplastically from the phloem throughout the tissues of the seed coat before unloading to the apoplast (symplastic pathway) (13,16,17,21). The symplastic unloading pathway has received the most support.The study of photosynthate unloading from legume seed coats has been advanced by the development of the "empty seed coat technique," in which the embryo of a developing seed is removed through an incision in the seed coat and a perfusion solution (or agar trap) is used to collect materials released from the seed coat for subsequent analysis (20,26,31). The use of the empty seed coat technique is complicated by the possibility that the surgical procedure, or the introduction of a solution into the seed coat "cup," may disturb phloem import into the seed coat and/or the process of unloading to the seed coat apoplast (15). Early studies using 'Supported by grant 87-CRCR-1-2397 from the U.S. Department of Agriculture (R.M.S.), grant DCB-8803837 from the National Science Foundation (R.T.), and funds provided by the Hatch Program.pulse-labeling of leaves (or petioles) indicated that the total amounts of labeled photosynthates imported to perfused and intact seeds were similar (20,26,31). However, quantitative comparisons of pulse-label accumulation by perfused versus intact seeds are unreliable when made over long time periods because the tracer fluxes produced by this technique are not sustained over time.Many problems inherent in pulse-labeling are circumvented by the use of steady-state labeling techniques. Steady-state labeling has been used for the quantitative analysis of various aspects of carbon partitioning in plants because the stable tracer fluxes produced by the continuous assimilation of "'CO2 at constant specific activity result in relatively simple trace...