Kinetics of 14 C-Photosynthate Uptake by Developing Soybean Fruit

Abstract

By pulse-labeling field-grown soybean leaves for 60 seconds at midday with "CO2 and then sequentially harvesting, dissecting, and extracting the radioactive fruiit tissues (of pod and seeds), the route, uptake kinetics, and metabolic fate of "C-photosynthate as it was imported by 35-to 40-day-old pods were determined. As the l14Clsucrose pulse entered the pods, the seeds became radioactive immediately but a lag of nearly 30 minutes occurred before label could be detected in the pod wail pericarp. Import of the 14C-pulse by the seeds was exclusively via the seed coats, where rapid unloading occurred. Maximum accumulation of label in the seed coat occurred in about 60 minutes at which time 59% of the total radioactivity in the fruit was in the three seed coats, whereas only 7% was in the cotyledons they enclosed. The photosynthate remained as l14CI-sucrose as it passed through the seed coat, but appeared to be hydrolyzed relatively soon after uptake by the cotyledons. By 2.5 hours, 60% of the 14C-photosynthate pulse had passed into the cotyledons with only 27% remaining in the seed coats. Inasmuch as there is no vascular connection between maternal seed coat and the developing embryo, cotyledonary uptake of sucrose released from the inner seed coat surface may require specialized transport mechanisms. In the very early stages of soybean seed growth, nourishment is obtained indirectly through extensive digestion of maternal ovule tissues. During this period the seeds enlarge slowly whereas the surrounding pod (ovary) grows rapidly (3). Soon, however, seeds utilize external photosynthate supplied via the pod. Biochemical studies of oil and protein deposition during soybean seed growth (1) indicate that large quantities of photosynthate are transported to the embryo and cotyledons as they develop. Import of photo-synthate necessary to maintain reported (4) rates of seed growth (3-8 mg dry weight seed-' day-'; [4]) can be as much as 7-20 mg sucrose seed-' day-' (calculated from ref. 14), approximately half of the sucrose production of each dm2 of illuminated leaf per day. Since the upper, more fully-illuminated leaves commonly supply several pods, some subtending and some at other nodes, photo-synthate availability may at times limit seed growth and productivity. Thus, although concurrent canopy photosynthesis is usually the primary source of photosynthate for seed growth (12, 15), redistribution of stored assimilates from pod walls (25) and stems (24) may contribute substantially in some cultivars. Anatomical studies on developing seeds in the Graminae have found specialized absorption structures in the region of seed attachment, where a discontinuity of phloem exists (27). Biochemical studies of the kinetics and mechanism of 14C-photosynthate uptake, however, leave considerable debate as to whether sucrose can be transported from the phloem into the developing endo-sperm in an unmodified form (16, 22). A similar lack of vascular continuity is known to exist between embryonic and maternal tissues of various legume species. including soybean (3, 20). While the literature contains studies of soybean phloem loading and transport kinetics (5, 23) and whole plant photosynthate distribution patterns (2), there is little information characterizing photo-synthate uptake and metabolism by the reproductive sink tissues of soybean (18, 19, 25) and none regarding the mechanisms involved. A thorough characterization of phloem unloading and photo-synthate uptake by developing soybean seeds is necessary to understand the mechanism of long distance translocation, and to determine if photosynthate availability may sometimes limit seed growth and productivity. The route and kinetics of '4C-photosyn-thate uptake by developing soybean seeds are reported here; short-term distribution and metabolism within fruit tissues are discussed, and findings are related to possible controls of photosynthate partitioning and seed productivity. MATERIALS AND METHODS Labeling Procedure. On selected plants within a population of field-grown Amsoy-71 soybeans (Glycine max (L.) Merr.), a single leaflet was photosynthetically labeled with 14CO2 (290 ,ul F-' CO2, 1.1% 02, 1.35 mCi mol' C02) shortly after solar noon on a clear day, approximately 35 days after flowering at that node. Radiant flux density (PAR), was 2.3 x 103 ,uE m-2 s'I as measured with a Licor-185 sensor. To reduce variability, replicate plants were selected for labeling that met the following requirements: between mainstem nodes 12 and 14 there was a recently fully expanded leaf, west-facing for uniform afternoon solar exposure, with a 32-cm petiole, three subtending pods at that node, each with three seeds 35-40 days of age (as determined by an external pod width measurement with calipers), and which was at least 2 m removed from other experimental plants. A 40-cm2 area in the center of the terminal leaflet was labeled for 60 s using a clip-on assimilation chamber built of Plexiglas with windows of IR-transparent polypropylene Propafilm Cl 10 (Imperial Chemical Industries, Ltd., Wilmington, Del.). With this chamber both abaxial and adaxial leaf surfaces were simultaneously exposed to 14CO2 supplied from a small lecture bottle. Plants were then allowed a "chase" period of varying lengths for photosynthesis and translocation. Recovery and Analysis of 4C-Photosynthate. Harvest intervals following labeling were chosen after a preliminary experiment with this system had identified the time of '4C arrival in the fruit. Harvested plants from the preliminary experiment were quickly cut into appropriate sections and frozen in liquid N2. Petioles were cut into 2-cm sections, frozen and later digested in 1.0 ml 70o HC104, 30o H202 (1:1, v/v) in capped scintillation vials at 60 C. The pods and seeds of this preliminary experiment were similarly digested. Digested samples were diluted with water and counted by liquid scintillation spectroscopy in Aquasol II cocktail (New 975