Relationship of endogenous abscisic Acid to sucrose level and seed growth rate of soybeans.

It has been proposed that abscisic acid (ABA) may stimulate sucrose transport into filling seeds of legumes, potentially regulating seed growth rate. The objective of this study was to determine whether the rate of dry matter accumulation in seeds of soybeans (Glycine max L.) is correlated with the endogenous levels of ABA and sucrose in those sinks. The levels of ABA and sucrose in seed tissues were compared in nine diverse Plant Introduction lines having seed growth rates ranging from 2.5 to 10.0 milligrams dry weight per seed per day. At 14 days after anthesis (DAA), seeds of all genotypes contained less than 2 micrograms of ABA per gram fresh weight. Levels of ABA increased rapidly, however, reaching maxima at 20 to 30 DAA, depending upon tissue type and genotype. ABA accumulated first in seed coats and then in embryos, and ABA maxima were higher in seed coats (8 to 20 micrograms per gram fresh weight) than in embryos (4 to 9 micrograms per gram fresh weight. From 30 to 50 DAA, ABA levels in both tissues decreased to less than 2 micrograms per gram fresh weight. Levels of sucrose were also low early in development, less than 10 milligrams per gram fresh weight at 14 DAA. However, by 30 DAA, sucrose levels in seed coats had increased to 20 milligrams per gram fresh weight and remained fairly constant for the remainder of the filling period. In contrast, sucrose accumulated in embryos throughout the filling period, reaching levels greater than 40 milligrams per gram fresh weight by 50 DAA. Correlation analyses indicated that the level of ABA in seed coats and embryos was not directly correlated to the level of sucrose measured in those tissues or to the rate of seed dry matter accumulation during the linear filling period. Rather, the ubiquitous pattern of ABA accumulation early in development appeared to coincide with water uptake and the rapid expansion of cotyledons occurring at that time. Whole tissue sucrose levels in embryos and seed coats, as well as sucrose levels in the embryo apoplast, were generally not correlated with the rate of dry matter accumulation. Thus, it appears that, in this set of diverse soybean genotypes, seed growth rate was not limited by endogenous concentrations of ABA or sucrose in reproductive tissues.

Effect of shading individual soybean reproductive structures on their abscisic Acid content, metabolism, and partitioning.

Pod set in soybean is related to carbon partitioning and may be, at least partially, regulated by abscisic acid (ABA) concentrations. The studies reported here examine the relationship between carbon and ABA partitioning, reproductive abscission and ABA metabolism. The partitioning of radiolabeled ABA and photoassimilates from leaves to flowers and endogenous ABA concentrations were determined in shaded and unshaded reproductive structures. Aluminum foil was gently placed over individual soybean reproductive structures for 48 hours at 0, 4, 12, 17, and 22 days after anthesis (DAA). Shading of flowers at 12, 17, and 22 DAA resulted in significantly reduced concentration of ABA. However, shading had no effect on the catabolism of exogenously supplied [(3)H] ABA. The shading treatment on the first four of the five dates reduced partitioning of photoassimilates and ABA from the subtending leaf to the flower. Shading of reproductive structures also caused a significant reduction in the amount of assimilate exported from the subtending leaf, at 17 DAA. We conclude that shade-induced premature reproductive abscission in soybean is not stimulated by high levels of ABA within reproductive structures, but that ABA may inhibit abscission of reproductive structures by playing a role in preferential assimilate partitioning.

Accumulation of C-radiolabel in leaves and fruits after injection of [C]tryptophan into seeds of soybean.

Injection of [(14)C]tryptophan into one seed in a soybean fruit resulted in recovery of radiolabel in a fraction that cochromatographed with indoleacetic acid (IAA) through three successive high performance liquid chromatography separations. Label was found in the putative IAA fraction in all of the fruit tissues sampled and in the blade of the leaf subtending the pod into which the radiolabeled tryptophan had been injected. This suggested that IAA or an IAA precursor was transported from seeds to other parts of the fruit and to subtending leaves.

Concentrations of Abscisic Acid and Indole-3-Acetic Acid in Soybean Seeds during Development.

Concentrations of abscisic acid (ABA) and indole-3-acetic acid (IAA) in seed parts were determined during reproductive development of soybean plants (Glycine max [L.] Merr. cv ;Chippewa 64'). The concentration of ABA and IAA changed independently in individual seed parts with time. Measurement of the level of ABA and IAA in whole seeds masked the changes which occurred in individual seed tissues. The concentration of ABA was generally highest and that of IAA was generally lowest in the embryonic axis of soybean seeds. In the testa, the IAA concentration was generally highest while the ABA concentration was generally the lowest compared to other parts of the seed.

Effects of pod removal on the transport and accumulation of abscisic Acid and indole-3-acetic Acid in soybean leaves.

Concentrations of abscisic acid (ABA) and indole-3-acetic acid (IAA) in the second most recently expanded trifoliolate leaf were determined during reproductive development of soybean (Glycine max [L.] Merr cv ;Chippewa 64'). The concentration of ABA in leaves was constant during most of the seed filling period until the seeds began to dry. The concentration of IAA in the leaves decreased throughout development. Removal of pods 36 hours prior to sampling resulted in increased concentrations of ABA in leaves during the period of rapid pod filling but had little effect on the concentration of IAA in leaves. ABA appears to accumulate in leaves after fruit removal only when fruits represent the major sink for photosynthate.ABA and IAA moving acropetally and basipetally in petioles of soybean were estimated using a phloem exudation technique. ABA was found to move mostly in the basipetal direction in petioles (away from laminae). IAA, primarily in the form of ester conjugate(s), was found to be moving acropetally (toward laminae) in petioles. The highest amount of IAA ester(s) was found in petiole exudate during the mid and late stages of seed filling. Removal of fruits 36 hours prior to exudation reduced the amount of IAA ester recovered in exudate, suggesting that fruits were a source of the IAA conjugate in petiole exudate.

Abscisic Acid and its relationship to seed filling in soybeans.

The effect of exogenous abscisic acid (ABA) on the rate of sucrose uptake by soybean (Glycine max L. Merr.) embryos was evaluated in an in vitro system. In addition, the concentrations of endogenous ABA in seeds of three soybean Plant Introduction (PI) lines, differing in seed size, were commpared to their seed growth rates. ABA (10(-7) molar) stimulated in vitro sucrose uptake in soybean (cv ;Clay') embryos removed from plants grown in a controlled environment chamber, but not in embryos removed from field-grown plants of the three PI lines. However, the concentration of ABA in seeds of the three field-grown PI lines correlated well with their in situ seed growth rates and in vitro [(14)C] sucrose uptake rates.Across genotypes, the concentration of ABA in seeds peaked at 8.5 micrograms per gram fresh weight, corresponding to the time of most rapid seed growth rate, and declined to 1.2 micrograms per gram at physiological maturity. Seeds of the large-seeded genotype maintained an ABA concentration at least 50% greater than that of the small-seeded genotype throughout the latter half of seed filling. A higher concentration of ABA was found in seed coats and cotyledons than in embryonic axes. Seed coats of the large-seeded genotype always had a higher concentration of ABA than seed coats of the small-seeded line. It is suggested that this higher concentration of ABA in seed coats of the large-seeded genotype stimulates sucrose unloading into the seed coat apoplast and that ABA in cotyledons may enhance sucrose uptake by the cotyledons.

Alteration of C-assimilate partitioning in leaves of soybeans having increased reproductive loads at one node.

The objectives of this study were to determine if the partitioning of recently fixed carbon between starch and water-soluble compounds could be altered by increasing the pod load in the leaf axil, and if the presence of source leaves acropetal to such a node would influence the partitioning of carbon within the subtending leaf. Soybeans (Glycine max L. Merr. cv Hodgson 78) were grown to full-bloom in a controlled environment chamber, and then deflowered at all nodes except the eighth. This treatment resulted in an 83% increase in the number of pods at the eighth node. At 24 days after flowering, one-half of the treated plants were girdled above the untreated node. Forty-two hours later, the eighth trifoliolate was pulsed with (14)CO(2) and sampled for radiolabeled starch and water-soluble compounds (WSC) at 0.5, 2, 4, 8, 12, and 24th after labeling.When no girdling was applied above the increased pod load at the eighth node more label was accumulated by the pod walls (+6.9%) and seeds (+6.3%) when compared to the controls. Starch accumulation was not altered in the labeled leaf of the nongirdled plants. When the stem was girdled above the eighth node, significantly less starch was retained in the labeled leaf. Girdling also resulted in an increase in label accumulation by the pod walls (+5.4%) and seeds (+6.6%). These data suggest that the plant will change the distribution patterns of assimilate to supply added sink demand before altering the partitioning of recently fixed carbon in the subtending leaf.

Effect of shortened photosynthetic period on C-assimilate translocation and partitioning in reproductive soyeans.

Starch accumulation rate in leaves of vegetative soybeans is inversely related to the length of the daily photosynthetic period. However, it is not known whether a similar response would be observed during reproductive growth. Soybeans (Glycine max L. Merr. cv Amsoy 71) were grown to three stages of reproductive growth (beginning seed, mid seed-fill, and late seed-fill) under 12-hour daylengths, and then shifted to 6-hour photosynthetic periods (12-hour photoperiods) for 4 days. One and 4 days after treatment, a mid-canopy leaf was pulsed with (14)CO(2), and sampled for radiolabeled starch and water-soluble compounds at 0.5, 1, 3, 9, and 21 hours after labeling.Plants exposed to the 6-hour photosynthetic periods at the beginning seed stage retained and incorporated significantly more label as starch than did those given 12-hour photosynthetic periods. However, plants exposed to the shortened photosynthetic periods at the late seed-fill stage partitioned less label into starch. Plants exposed at mid seed-fill gave a variable response.Shortened photosynthetic periods resulted in preferential partitioning of recently fixed carbon to the seed at the expense of the pod wall. The results of these experiments suggest that the increased sink demand present during late reproductive growth may be of greater importance in control of leaf starch accumulation than is the length of the daily photosynthetic period.

Source/Sink relations of abscising and nonabscising soybean flowers.

The partitioning of recently fixed (14)C to setting and abscising flowers within the axillary raceme of ;Clark' isoline E1t soybeans (Glycine max L. Merr) was examined as a function of time after anthesis of individual flowers. In such racemes, the first four flowers showed a 17% abscission while the next four flowers showed 47% abscission.Source/sink relations of flowers I-IV (normally setting) were compared to those of flowers V-VIII (normally abscising) by pulse labeling source leaves with (14)CO(2) and determining the radioactivity of individual flowers after a 4-hour chase period. The relative specific activity (RSA;% disintegrations per minute per% dry weight), sink strength (% disintegrations per minute), and its components, sink size (milligrams dry weight) and sink intensity (% disintegrations per milligram dry weight) were then calculated as a function of days after anthesis.Sink intensity (i.e. the competitive ability to accumulate photoassimilate per unit mass) was very high prior to anthesis of both setting and abscising flowers. Sink intensity then became very low for the first 3 days following anthesis after which it recovered in normally setting flowers, but failed to recover in normally abscising flowers. It is concluded that soybean reproductive abscission is determined at or very near the day of anthesis.

Light and Shade Effects on Abscission and C-Photoassimilate Partitioning among Reproductive Structures in Soybean.

Field experiments were conducted in 1981 and 1982 to study the effects of low-irradiance supplemental light on soybean (Glycine max [L.] Merr. cv Evans) flower and pod abscission. Cool-white and red fluorescent lights illuminated the lower part of the soybean canopy during daylight hours for 3 weeks late in flowering. At the same time, flowers and young pods on half the plants were shaded with aluminum foil. Flowers were tagged at anthesis and monitored through abscission or pod maturity.Responses to red and white lights were similar. Supplemental light tended to reduce abscission and increase seed weight per node compared to natural light. Shading flowers and pods increased abscission and reduced seed weight per node. Number of flowers produced per node, individual seed weight, and seeds per pod were not affected by light or shade treatments.Further studies examined the effects of shading reproductive structures on their capacity to accumulate (14)C-photoassimilates. Individual leaves were pulse labeled with (14)CO(2) 1, 2, and 4 weeks post anthesis. Flowers and pods in the axil of the labeled leaf were covered with aluminum foil 0, 24, 72, and 120 hours before pulsing.Shading flowers and pods resulted in a 30% reduction in the relative amount of radiolabel accumulated from the source leaf. The reduction in (14)C accumulation due to shading was evident regardless of the length of the shading period and was most pronounced when the shades were applied early in reproductive development. We conclude that light perceived by soybean flowers and young pods has a role in regulating both their abscission and their capacity to accumulate photoassimilates.

Ontogenetic variation of four cytokinins in soybean root pressure exudate.

Cytokinins exported from the root may be involved in the correlative control of plant development. To test this hypothesis in soybean ((Glycine max [L.] Merr. cv. McCall, cv Chippewa 64, and cv Hodgson 78), cytokinins were intercepted en route from the root to the shoot by collecting root pressure exudate from detopped roots. The quantities of four cytokinins in the exudate were studied throughout the development of plants grown in the field and in controlled environment chambers. Zeatin, zeatin riboside, and their dihydro derivatives, dihydrozeatin and dihydrozeatin riboside, were isolated and quantitated using high-performance liquid chromatography.Cytokinin fluxes (pmoles per plant per hour) were independent of exudate flux (grams per plant per hour). All fluxes are averages for a 6- or 8-h collection period. The ribosides accounted for the majority of the observed cytokinin transport. The fluxes of zeatin riboside and dihydrozeatin riboside increased from low levels during vegetative growth to maxima during late flowering or early pod formation. Before the seeds began rapid dry matter accumulation, zeatin riboside and dihydrozeatin riboside fluxes decreased and remained at low levels through maturation. The fluxes of zeatin and dihydrozeatin were low throughout development.No correlation was found between cytokinin fluxes and nodule dry weight or specific nodule activity (acetylene reduction).The timing of distinct peaks in zeatin riboside and dihydrozeatin riboside fluxes during flowering or pod formation suggests that cytokinins exported from the root may function in the regulation of reproductive growth in soybean.

Effect of Atmospheric CO(2) Enrichment on Growth, Nonstructural Carbohydrate Content, and Root Nodule Activity in Soybean.

The objective of this study was to determine whether the supply of current photosynthate was limiting root nodule activity. Both short-term (36 hours) and long-term (16 days) periods of CO(2) enrichment were imposed on vegetative, growth chamber-grown soybean plants (Glycine max. [L.] Merr. cv. ;Clay') to increase the supply of current photosynthate and to observe the effects on photosynthate partitioning in the plants, plant growth, and root nodule activity.Neither total nor specific nodule activities were increased during exposure to short-term (36 hours) CO(2) enrichment. Dry weight of the leaves increased after 12, 24, and 36 hours of CO(2) enrichment and dry weight of the stems plus petioles increased after 36 hours of CO(2) enrichment. Dry weights of the roots and nodules were not altered by short-term CO(2) enrichment. Short-term CO(2) enrichment increased the total nonstructural carbohydrates in the leaves and stems plus petioles, but not in the roots and nodules. Analyses of the separate pools of carbohydrate reserves indicated that the majority of the additional carbohydrate provided by short-term CO(2) enrichment was stored as leaf starch with relatively little being partitioned to the roots and nodules.Long-term CO(2) enrichment (16 days) did not enhance specific nodule activity. Shoot, root, and nodule dry weights were increased 109, 34%, and 56% respectively. Total nodule activity per plant was significantly enhanced only after 16 days of treatment and was related to increased nodule mass. These results indicate that the increased total nodule activity in response to CO(2) enrichment is a consequence of a general growth response of the plant.Results of both studies indicate that nodule activity was not directly limited by current photosynthesis but rather by the partitioning and utilization of photosynthate in the plant.

Identification of a dihydrophaseic Acid aldopyranoside from soybean tissue.

A previously unidentified abscisic acid metabolite has been isolated and characterized. (+/-)-[2-(14)C]Abscisic acid was incubated in intact soybean leaves and pods; the radiolabeled metabolite was purified by high performance liquid chromatography with on-line scintillation spectrometry detection. Gas chromatography-mass spectrometry was used to obtain spectra of the acetylated and methyl esterified derivatives. The data were consistent with a proposed dihydrophaseic acid-aldopyranoside identity. Conjugation through the 4'-hydroxyl of dihydrophaseic acid is suggested.

Abscisic Acid Translocation and Metabolism in Soybeans following Depodding and Petiole Girdling Treatments.

It was found earlier that depodding and girdling treatments which obstruct translocation, result in increased leaf AbA levels and partial stomatal closure. In the present work (+/-) [2-(14)C]abscisic acid (AbA) was introduced into leaves and the mass, and radioactivity of AbA and AbA-metabolites were analyzed following translocation obstruction to determine whether the increased AbA was due to higher rates of synthesis, or lower rates of catabolism or export. The (+/-) [2-(14)C]AbA was introduced into soybean (Merr.) leaves by injection into the petiole region. AbA and AbA-metabolites (phaseic acid [PA], dihydrophaseic acid [DPA], AbA-conjugate, and an unknown metabolite) were separated with preparative high performance liquid chromatography. Methyl esters of AbA (free and that released after hydrolysis of AbA-conjugate), PA and DPA were determined with gas chromatography using electron capture detection.The level of AbA in leaf blades increased after girdling or depodding as was found previously. Accompanying this was an increase in PA in girdled leaves; but no discernible trends in the levels of DPA and AbA-conjugate were evident. The (+/-) [2-(14)C]AbA specific radioactivities declined similarly for all treatments, indicating that these treatments did not increase the rate of AbA synthesis.Export of injected (+/-)[2-(14)C]AbA from leaves was substantial. After girdling or depodding, this export was obstructed, as evidenced by the lack of decline in leaf blade radioactivity or lack of increase in pod radioactivity following these treatments. The higher AbA levels, which were observed in leaf blades of girdled and depodded plants, could be attributed largely to the translocation obstruction.

Effect of obstructed translocation on leaf abscisic Acid, and associated stomatal closure and photosynthesis decline.

Pod removal or petiole girdling, which causes obstruction of translocation, was found in our previous study to cause reduced rates of photosynthesis in soybean leaves due to stomatal closure. The purpose of this research was to determine the involvement of photoassimilate accumulation and leaf abscisic acid (ABA) levels in the mechanism of stomatal closure induced by such treatments.Leaf glucose and sucrose levels increased during the initial 12-hour period after depodding or petiole girdling. Starch, which represents a much larger pool of leaf carbohydrate, was not perceptibly increased above control levels during the 12-hour posttreatment period.When leaflets were exposed to nonphotosynthetic environments (shading or CO(2)-free air) for a 24-hour period after the translocation-obstructing treatments were applied and then returned to normal light or CO(2) concentration, stomatal diffusive conductivity was reduced 65% and 85% with depodding and girdling, respectively. These reductions were comparable to those previously observed without an intervening nonphotosynthetic exposure, thus indicating that photosynthate accumulations were not necessary for the observed response.Free and bound ABA (released on alkaline hydrolysis) were determined by gas liquid chromatography with electron capture detection following preparative high performance liquid chromatography. Free ABA in monitored leaves increased almost 10-fold 48 hours after complete depodding and 25-fold 24 hours after petiole girdling of such leaves. By 3 hours after treatment, in a time course study, free ABA had increased 2-fold above control values in depodded and 5-fold in girdled leaves. Leaf concentrations of bound ABA did not appear to be related to the treatment effects on stomata.Thus, the translocation-obstructing treatments cause an increased level of ABA by a mechanism not involving accumulation of photoassimilate. Increased leaf ABA levels, which were independent of water stress or leaf water potential, appear to be involved in the stomatal closure response. It is suggested that the mechanism of increased leaf ABA levels following translocation-obstruction may be due to an interference with normal translocation of ABA out of leaves.

Stomatal closure and photosynthetic inhibition in soybean leaves induced by petiole girdling and pod removal.

The presence of strong sinks of photoassimilates is thought to stimulate photosynthesis by minimizing photosynthetic end product accumulation in leaves. This hypothesis was examined in soybeans (Glycine max [L] Merr.) with treatments designed to alter the phloem translocation of photoassimilates out of source leaves. Pod removal and petiole girdling resulted in 70 and 90% reductions, respectively, in leaf CO(2) exchange rate. Reductions of similar magnitude also were observed in stomatal diffusive conductivity.Time course data showed that CO(2) exchange rates as well as stomatal conductivities were significantly reduced 0.5 hour after girdling and 5 hours after pod removal, thus suggesting that stomatal closure was the cause of the reduced rates of photosynthesis.Mesophyll conductivities to CO(2), calculated from gas exchange data, were not affected by the treatments. Radiocarbon assimilation rates by leaf slices floated in KH(14)CO(3) solutions were not reduced by pod removal and were reduced only 10% by girdling. It was concluded that treatments which block or slow translocation from source leaves reduce their photosynthetic rate by inducing stomatal closure.

Effects of Sink Removal on Photosynthesis and Senescence in Leaves of Soybean (Glycine max L.) Plants.

Photosynthetic rate, ribulose 1,5-bisphosphate carboxylase activity, specific leaf weight, and leaf concentrations of carbohydrates, proteins, chlorophyll, and inorganic phosphate were determined periodically from midbloom until maturity in leaves of soybean plants (Glycine max L., var. Hodgson) from which reproductive and vegetative sinks had been removed 32 hours before measurement, or continuously since midbloom.Leaf photosynthesis, measured in the top of the canopy, was partially inhibited by both sink removal treatments. This inhibition was of constant magnitude from midbloom until maturity.Leaf photosynthesis in the top of the canopy declined from midbloom until maturity in the control as well as in the desinked plants. The decline in photosynthesis was gradual at first, but later became more abrupt. The photosynthetic decline was equally evident in the yellowing leaves of control plants and in the dark green leaves of the continuously desinked plants.Neither the inhibition of photosynthesis by sink removal nor the decline in photosynthetic rate with time was clearly related to any of the measured traits.

Rapid separation and quantification of abscisic Acid from plant tissues using high performance liquid chromatography.

Abscisic acid (ABA) was purified from soybean (Glycine max [L.]) seed extract using a preparative high performance liquid chromatography (HPLC) procedure. The preparative procedure was rapid (70 minutes per sample), required no prior partitioning for purification and was quantitative as demonstrated with an internal standard of [2-(14)C]ABA, of which 98.9% was recovered.Following purification by the preparative HPLC procedure, the ABA in a soybean seed extract was quantified using either GLC with an electron capture detector (GLC-EC) or by analytical HPLC with a UV detector. For soybean seed extracts, two analytical HPLC column packing materials were found adequate: muPorasil and muBondapak-NH(2) (Waters Associates). However, with complex tissue extracts, such as soybean leaf and nodule tissues, only GLC-EC had the necessary selectivity and sensitivity.

Carbon dioxide compensation points in related plant species.

Both high and low C0(2) compensation concentrations were found in the plant genera-Panicum, Cyperus, and Euphorbia. Within each genus, however, high and low compensations were found in different subgenera. Thus, they may not be genetically closely related. No significant differences in CO(2) compensation were found among 100 genetic lines of Triticum aestivum L. or among 20 lines of Hordeum vulgare L.

Stomatal density and responsiveness of banana fruit stomates.

Determination of stomatal densities of the banana peel (Musa acuminata L. var Hort. Valery) by microscopic observations showed 30 times fewer stomates on fruit epidermis than found on the banana leaf. Observations also showed that peel stomates were not laid down in a linear pattern as on the leaf.It was demonstrated that stomatal responses occurred in banana fruit. Specific conditions of high humidity and light were necessary for stomatal opening: low humidity and darkness were necessary for closure. Responsiveness of the stomates continued for a considerable length of time after the fruit had been severed from the host.