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Leaf starch accumulation and seed set at phloem-isolated nodes in soybean
Field Crops Research 68 (2000) 113±120
Leaf starch accumulation and seed set at phloem-isolated nodes in soybean W.P. Bruening*, D.B. Egli Department of Agronomy, University of Kentucky, Lexington, KY 40546-0091, USA Received 20 April 2000; received in revised form 8 July 2000; accepted 11 July 2000
Abstract There is usually a linear relationship between pod and seed number per unit area and assimilate supply in soybean (Glycine max L. Merrill) communities. In contrast, the relationship is curvilinear at single phloem-isolated nodes. To further investigate this curvilinear relationship, we evaluated photosynthesis, leaf starch levels and pod and seed number at phloem-isolated nodes of cultivar Elgin 87 in three greenhouse experiments. The main stem was girdled between the ®fth and sixth nodes (uni®oliolate node was node one) when the ®rst ¯owers opened at the sixth node. The main stem above the sixth node was removed and defoliation (®ve or six levels from 0 to 100%) created a range in assimilate supply. Girdling increased leaf starch levels 3- to 7-fold over non-girdled plants within 7 days. In two experiments starch decreased to control levels within 14±27 days after girdling, but in a third experiment the increase was maintained for 28 days. Defoliation reduced leaf starch levels, with 66±91% defoliation lowering it to the level in the non-girdled controls. Pod and seed number were directly related to assimilate supplies at low levels of assimilate availability when there was no accumulation of starch. There were only relatively small increases in pod and seed number at high levels of assimilate availability, but there were large accumulations of starch in the leaves. Flower and pod abortion was always above 50%, so pod set was not limited by ¯ower availability. The failure of pod and seed number to respond to high levels of assimilate availability suggests that there may be other processes involved in determining pod and seed number at isolated nodes in soybean. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Leaf starch accumulation; Phloem-isolated nodes; Soybean; Defoliation; Girdling
1. Introduction The number of seeds per unit area is usually related to canopy photosynthesis during some critical stage of reproductive growth in most grain crops. For example, modi®cation of canopy photosynthesis or crop growth rate of soybean by shade (Egli and Zhen-wen, 1991; Egli, 1993), CO2 enrichment (Jones et al., 1984) or increased irradiance (Schou et al., 1978) caused cor*
Corresponding author. Tel.: 1-859-257-9404; fax: 1-859-257-7874. E-mail address: [email protected] (W.P. Bruening).
responding changes in pod and seed number. Similar responses have been reported for other crops (Fischer, 1985; Kiniry and Knievel, 1995). Varying the leaf area at a single phloem-isolated soybean node, created by girdling the main stem, demonstrated that increasing total photosynthesis or nodal carbon input (NCI) above a minimum level did not substantially increase pod or seed number in spite of 30±70% reproductive abortion (Bruening and Egli, 1999). Maximum pod and seed number frequently occurred at less than 50% of maximum NCI in two ®eld experiments with three cultivars. Similar curvilinear relationships were reported for individual plants of
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W.P. Bruening, D.B. Egli / Field Crops Research 68 (2000) 113±120
maize (Zea mays L.) and sun¯ower (Helianthus annuus L.) (Edmeades and Daynard, 1979; Vega et al., 1998). This curvilinear response at isolated nodes in soybean contrasts with the relationship at the community level, where seed number is usually linearly related to crop growth rate (an estimate of canopy photosynthesis) during ¯owering and podset (Ramseur et al., 1985; Charles-Edwards et al., 1986; Egli, 1993). The physiological basis for this difference between phloem-isolated nodes and plant communities is not known. Some of the carbon ®xed in photosynthesis may be converted to starch in the leaf and not immediately transported to developing sinks (Preiss and Sivak, 1996) suggesting that simple estimates of photosynthesis may not adequately describe the assimilate supply to developing reproductive structures. The amount of starch in a soybean leaf responds quickly to changes in source±sink ratios. For example, reducing sink size increased leaf starch content and decreased carbon exchange rate (CER) (Mondal et al., 1978; Miceli et al., 1995). Alternatively, decreasing the source±sink ratio by shading all but a few leaves decreased leaf starch content and/or increased CER of unshaded leaves for at least 8 (Thorne and Koller, 1974) or 22 (Peet and Kramer, 1980) days after treatment. Petiole girdling to block phloem export in soybean dramatically increased leaf starch content and reduced leaf CER for at least 7 days (Goldschmidt and Huber, 1992), however, over shorter intervals (between 0.5 and 12 h) leaf CER decreased, but there was no signi®cant effect on leaf starch content (Setter and Brun, 1980). The availability of assimilate to the reproductive sink is related to CER in the leaf and changes in leaf carbohydrate levels. Evaluation of changes in leaf starch and soluble carbohydrates at phloem-isolated nodes in soybean will contribute to a better understanding of the relationship between photosynthesis and pod and seed number. Thus, the objective of this study was to evaluate the relationship between leaf photosynthesis, starch content and pod and seed number at phloem-isolated nodes in soybean. 2. Materials and methods Three greenhouse experiments were conducted at the University of Kentucky during 1996 and 1998.
Seeds of soybean cultivar Elgin 87 (Maturity Group II) were planted in 3 l pots ®lled with a 2:1 (v/v) mixture of silt loam soil and vermiculite on 23 April 1996 (Exp. 1), 30 August 1996 (Exp. 2), and 16 July 1998 (Exp. 3). Pots were overseeded and thinned to one plant per pot after emergence. The plants were fertilized with 0.3 g per pot of Peters Professional fertilizer (20-20-20) at approximately weekly intervals. On 4 June 1996 (Exp. 1), 11 October 1996 (Exp. 2) and 18 August 1998 (Exp. 3) the sixth node (unifoliolate node was node one) on the main stem of each plant was isolated by girdling the stem below the node and removing the portion of the plant above the node. The stem was girdled when the ®rst ¯owers at the girdled node opened and the plants were at growth stage R1 (Fehr and Caviness, 1977). Girdling was accomplished by electrically heating a bare constantan wire (approximately 0.7 mm diameter) wrapped around the stem as described previously (Bruening and Egli, 1999). The girdle was considered a success if, after 2 days, a complete band of dead tissue was observed around the stem. Variation in assimilate production at the girdled nodes was created by removing 0 (non-defoliated), approximately 34 (middle lea¯et removed), 66 (two side lea¯ets removed), 83 (two side lea¯ets and half of middle lea¯et removed), 91 [two side lea¯ets and 75% of the middle lea¯et removed (Exp. 3 only)], and 100% (entire leaf removed) of the leaf area when the plants were girdled. To reduce assimilate supply by shading, plants from the non-defoliated treatment in Exp. 3 were placed under 63% shade cloth when they were girdled. Leaf area of the center and side lea¯ets removed from plants receiving the 100% defoliation treatment was determined to estimate the leaf area removed in the other defoliation treatments (Bruening and Egli, 1999). A control±cut treatment (non-girdled, non-defoliated plant with the main stem above the sixth node removed) was included to provide a basis of comparison of the effect of girdling on photosynthesis and leaf starch content. The plant above the girdled node was removed on all treatments, thus they were all equally exposed to any possible effect of removing the stem apex. A completely randomized experiment design was used with 6±15 replications (plants) per treatment. Girdling was not always successful resulting in a variable number of plants per treatment.
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After girdling and defoliation, leaf CER was measured in Exp. 2 and 3 with a LI-6400 portable photosynthesis system (Li-Cor, Inc., Lincoln, NE). The CER of four leaves per treatment was determined before girdling and at approximately 5-day intervals on relatively sunny days when photosynthetically active radiation (PAR) was generally greater than 1000 mmols mÿ2 sÿ1 (Exp. 2) between 1100 and 1400 h. In Exp. 3, leaves were exposed to a constant PAR of 1500 mmols mÿ2 sÿ1 during the CER determination with a Li-Cor 6400-02B LED Red/Blue Light Source. The total daily photosynthesis of the leaf determines the daily carbon input (DCI) at the girdled node. Photosynthesis in a well watered greenhouse environment should be determined primarily by PAR, air temperature and the inherent photosynthetic capacity of the leaf. Thus, daily CER could be estimated with a model that accounts for these variables. The PAR and temperature in the greenhouse were measured at the top of the plants and averaged over 0.5 h intervals with a Li-Cor 1000 data logger. A PAR response curve, created by measuring CER at 13 PAR levels from 50 to 2000 mmol mÿ2 sÿ1 on two replications of leaves from Elgin 87 plants (control±cut) in the greenhouse, was used to estimate CER from measured PAR levels. Regression analysis of the relative CER (where, Y CER expressed as a percent of CER at 1500 mmol mÿ2 sÿ1 PAR) resulted in Eq. (1) (R2 0:99, regression was signi®cant at P 0:0001). Y ÿ107:7087 28:4266 ln PAR
(1)
The CER for each of the six defoliation treatments for each 0.5 h period during the day was estimated by ®rst calculating Y from the measured PAR (Eq. (1)) and then converting it to CER by multiplying Y by the CER measured frequently at 1500 mmol mÿ2 sÿ1 (CER1500) for each treatment. Daily values of CER1500 were estimated by linear interpolation between the periodic estimates (four over the 14day period). Bruening and Egli (1999) demonstrated that defoliation of leaves at phloem isolated nodes affected CER, making these adjustments for treatment effects necessary. The estimated CER was adjusted for temperature using a CER-temperature response curve from Norman and Arkebauer (1991), however, day time greenhouse temperatures were usually in the range (26±368C) where their curve predicts little
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response to temperature. Average maximum PAR over the 14-day period was 1256 mmol mÿ2 sÿ1 with a range from 774 to 1423 mmol mÿ2 sÿ1. Total daily CER was multiplied by leaf area per node to get DCI per node. The root mean square error comparing predicted CER with CER determined at a range of ambient PAR levels was 2.1 mmols CO2 mÿ2 sÿ1 (n 13), thus, the estimated DCI provided an adequate representation of the effect of daily and diurnal variation in PAR and temperature on total CER. Lea¯ets at the girdled node were harvested between 1100 and 1400 h at approximately 7- (Exp. 1, 3 replications) or 5-day intervals (Exp. 2 and 3, 4 replications) for carbohydrate analysis. Leaves were placed on ice in the greenhouse, taken immediately to the laboratory where the leaf area was determined before they were frozen, freeze dried and ground for analysis after the dry weight was determined. Total extractable sugar and starch contents were determined using the techniques of Heberer et al. (1985). At maturity, the number of pods, seeds, and ¯ower scars (Jiang and Egli, 1993) at the girdled node was determined. The combined ¯ower and pod abortion was calculated from the number of ¯owers and mature pods. 3. Results and discussion There was a curvilinear relationship between DCI at the girdled node and pod and seed number in Exp. 3 (Fig. 1). Pod and seed number at the girdled node reached near maximum levels at relatively low DCI levels (<4 mmol CO2 per node per day). Similar curvilinear relationships were found in Exps. 1 and 2 and in ®eld experiments with this cultivar and two other cultivars (Bruening and Egli, 1999). Combined ¯ower and pod abortion was substantial at the high DCI levels (i.e. low levels of defoliation) in these greenhouse experiments (51±74%) (Table 1) and in previous ®eld experiments (32±71%) (Bruening and Egli, 1999), suggesting that the curvilinear response did not result from a lack of ¯owers. Defoliation had no signi®cant effect on the total number of ¯owers at the girdled node (data not shown). Defoliation had almost no signi®cant effect on seeds per pod (Table 1), so the variation in seeds per node was primarily determined by the fate of the developing pod. In
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generally accepted concept that the number of reproductive structures is directly related to assimilate supply (Stephenson, 1981; Charles-Edwards et al., 1986; Egli, 1998). Since the supply of assimilate to a developing reproductive structure is not necessarily equivalent to leaf CER, we decided to investigate the relationship between soluble sugar and starch levels in the leaf, and pod and seed set at phloem-isolated nodes. Starch levels in leaves at phloem-isolated nodes in all three experiments increased rapidly and signi®cantly (P 0:05) after girdling, reaching maximum levels within 6±7 days (Fig. 2). There was no change in starch levels in leaves from the non-girdled control plants in Exp. 1 and 2, but in Exp. 3 starch increased and there was no signi®cant difference between leaves from the control and girdled plants until the fourth sample (13 days after girdling). The difference between the control and girdled plants decreased with time in Exps 1 and 3, but in Exp. 2 girdled plants maintained high starch levels for nearly 28 days. Leaf starch levels in soybean respond quickly to alterations in source±sink ratios (Shibles et al., 1987; Egli, 1999), so the increase in leaf starch reported here probably re¯ects a sink limitation imposed by girdling. Similar starch accumulations after girdling have been reported for soybean (Goldschmidt and Huber, 1992), wheat (Triticum aestivum L.) (MacKown and VanSanford, 1986), pea (Pisum sativum L.) and cotton (Gossypium hirsutum L.) (Goldschmidt and Huber, 1992). Defoliation at the girdled node reduced total photosynthesis per node (Bruening, 1997; Bruening and
Fig. 1. The relationship between assimilate supply and pod and seed number at phloem-isolated nodes in soybean, cultivar Elgin 87, Exp. 3. The daily carbon input was averaged over the ®rst 14 days after the plants were girdled. The symbols , identify the 63% shade treatment. The regression analysis with pod and seed number was signi®cant at P 0:01. The bars represent standard error of the mean.
contrast, there was variation in seeds per pod among experiments with the plants in Exp. 3 producing, on the average, 44% more than plants in Exp. 1 and 2 (Table 1). The basis for this differential response is not known. As reported previously (Bruening and Egli, 1999), the failure of pod and seed number to increase rapidly at high DCI levels is obviously not consistent with the
Table 1 Effect of defoliation at phloem isolated nodes on seeds per pod and reproductive abortion in three greenhouse experiments, cultivar Elgin 87 Experiment
Defoliation (%) 0
Seeds per pod 1 2 3a
1.7 1.7 2.4
Flower and pod abortion (%) 1 58 2 74 3b 51 a b
LSD (0.05) 33 1.8 1.8 2.8 61 73 61
66 1.8 2.1 2.7 61 70 72
The 63% shade-non-defoliated treatment had 2.7 seeds per pod. The 63% shade-non-defoliated treatment had 55% abortion.
83 1.8 1.8 2.6 69 72 72
91 ± ± 2.5 ± ± 80
NS NS 0.3 10 NS 10
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Fig. 2. The effect of girdling the main stem to block phloem transport on starch levels (g mÿ2 leaf area) in the leaf at the girdled node in three greenhouse experiments. The bars represent the LSD (P 0:05) for comparison of treatments at any sampling date.
Fig. 3. The effect of defoliation and shade on starch levels (g mÿ2 leaf area) in leaves at the girdled node in three greenhouse experiments. The bars represent the LSD (P 0:05) for comparison of treatments at any sampling date.
Egli, 1999) and signi®cantly (P 0:05) reduced the accumulation of leaf starch in all three experiments (Fig. 3). These reductions were generally in proportion to the degree of defoliation, except in Exp. 3 where there were no consistent differences among the 66, 83, and 91% defoliation treatments. Shading (63%) nondefoliated plants in Exp. 3 reduced starch to levels found in the highest defoliation treatments. Starch levels at the highest levels of defoliation in all experiments were generally similar to or below levels in ungirdled control plants (Figs. 2 and 3). Reducing photosynthate production per node by defoliation apparently reduced the source±sink imbalance created by girdling and reduced starch accumulation in the
leaves, a common response to reduced source activity (Huber et al., 1984; Shibles et al., 1987; Egli, 1999). Rather large reductions in DCI (66% defoliation) were required to reduce starch levels to those found in non-girdled plants, suggesting that approximately half of the assimilate was translocated away from the node subtending the sixth leaf (i.e. the node that was girdled) in control plants. Soluble sugars were not in¯uenced by defoliation in Exp. 2 and 3, but in Exp. 1 the soluble sugar levels followed the changes in starch (Fig. 4). There was no signi®cant difference in soluble sugar levels between leaves on control (non-girdled) and non-defoliated girdled plants, except for the 7-day sample in Exp. 1
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the response curve de®ned by the defoliation treatments (Fig. 1). Excessive accumulation of starch is an indicator of a sink limited system, i.e. the supply of assimilate exceeds the capacity of the sinks (Shibles et al., 1987). Leaf starch accumulated at high DCI levels (e.g., defoliation 66%, Fig. 3), but in spite of this apparent excess assimilate supply, there were only small increases in pod and seed number. The relatively large reductions in pod and seed number as the DCI decreased below 4 mmol CO2 per node per day (defoliation > 66%) in Exp. 3 (Fig. 1) were associated with minimal changes in average leaf starch levels (4.4±5.4 g mÿ2, Fig. 5). However, the relatively small changes in pod and seed number at higher DCI levels in all three experiments were associated with large increases in average leaf starch, starting at approximately 5 g mÿ2. This bi-phasic relationship between
Fig. 4. The effect of defoliation and shade on soluble sugar levels (g mÿ2 leaf area) in leaves at the girdled node in three greenhouse experiments. The bars represent the LSD (P 0:05) for comparison of treatments at any sampling date.
(data not shown). Thus, girdling and defoliation affected starch levels without consistently affecting soluble sugars. In contrast, shading non-defoliated plants reduced both starch and sugar levels in Exp. 3, although the effect on starch was much larger that the effect on soluble sugars (Figs. 3 and 4). Apparently altering source±sink ratios by reducing photosynthesis per unit area with shade elicited a different response in soluble sugar levels than reducing total photosynthesis per node by defoliation. This differential response did not seem to in¯uence the relationship between DCI and pod and seed number. The data from the shade treatment are consistent with
Fig. 5. The relationship between average leaf starch levels (g mÿ2 leaf area) for all samples from 1 to 14 days after girdling and pods and seed per node on girdled plants in three greenhouse experiments.
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starch and pod and seed number illustrates the apparent change in sink limitations that occurred as assimilate availability increased. Below approximately 66% defoliation, pod and seed number were closely associated with assimilate availability and there were minimal changes in leaf starch levels. The starch level in these leaves (5 g mÿ2) was similar to those found in non-girdled plants, suggesting that non-girdled plants may also exhibit a close association between pod and seed number and assimilate supplies. This biphasic relationship was not evident in Exps. 1 and 2 because the defoliation treatments did not reduce DCI and leaf starch to low enough levels (Fig. 5). Apparently when the assimilate supply increased to a critical level, pod set stopped and the leaves began to accumulate starch, re¯ecting a sink limitation. This part of the bi-phasic response, which is dif®cult to understand given the high level of ¯ower and pod abortion, may be related to the timing of ¯owering and pod development. The ®rst ¯owers and pods to develop may not initially provide a large enough sink to use all of the assimilate, resulting in starch accumulation. However, the assimilate available to ¯owers and pods developing later may be limited when the seeds in these early pods enter the rapid growth phase and consume relatively large amounts of assimilate. This lack of assimilate may cause late developing ¯owers and pods to abort, as we proposed earlier (Bruening and Egli, 1999). The eventual decline in leaf starch levels in Exp. 1 and 3 (Fig. 2) is consistent with this scenario. However, such declines in starch did not occur in Exp. 2, raising the possibility that late pod development may have also been prevented by a dominance type mechanism, possibly involving hormones (Bangerth, 1989). Clearly, much remains to be learned about the mechanisms regulating pod and seed set in soybean. The relationship between average leaf starch levels and pods per node was similar across experiments (Fig. 5), but there were large differences among experiments in seed number per node. The plants in Exp. 3 produced more seeds per node and per pod (Table 1) than the plants in Exp. 1 at the same starch level. The reason for these differences is not clear, but the results emphasize that both pod and seed set are involved in the determination of seed number. Pod number is determined by the number of ¯owers and the proportion that produce mature pods. Individual
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seeds in a pod may also develop or abort and seed number is the end result of these two processes. Abortion of pods probably occurs ®rst with seed abortion occurring only after the pod is fully developed. The variation in Fig. 5 may re¯ect an interaction between the greenhouse environment and this difference in occurrence of the critical growth stage. Seed number was also more responsive to higher DCI levels than pod numbers in Exp. 3 (Figs. 1 and 5), further evidence of a possible environmental interaction. In summary, pod and seed number at phloem-isolated nodes was only closely related to assimilate supplies at relatively low levels of assimilate availability. Pod and seed number exhibited only small increases at high levels of assimilate availability, but there were large accumulations of starch in the leaves, indicating that pod and seed number was not limited by assimilate supply. The failure of pod and seed number to respond to the greater assimilate availability suggests that there may be other processes, in addition to assimilate supply, involved in determining pod and seed number at isolated nodes in soybean. The exact nature of these processes remains to be determined. Acknowledgements Paper no. 00-06-62. Published with the approval of the Director of the Kentucky Agricultural Experiment Station, University of Kentucky, Lexington. References Bangerth, F., 1989. Dominance among fruits/sinks and the search for a correlative signal. Physiol. Plant 76, 608±614. Bruening, W.P., 1997. The relationship between photosynthesis and seed number in soybean cultivars with differences in seed size. M.S. Thesis, University of Kentucky, Lexington, KY. Bruening, W.P., Egli, D.B., 1999. Relationship between photosynthesis and seed number at phloem isolated nodes in soybean. Crop Sci. 39, 1769±1775. Charles-Edwards, D.A., Doley, D., Rimmington, G.M., 1986. Modeling Plant Growth and Development. Academic Press, Sydney, Australia, 235 pp. Edmeades, G.O., Daynard, T.B., 1979. The relationship between ®nal yield and photosynthesis at ¯owering in individual maize plants. Canad. J. Plant Sci. 89, 585±601. Egli, D.B., 1993. Cultivar maturity and potential yield of soybean. Field Crops Res. 32, 147±158.
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Egli, D.B., 1998. Seed Biology and the Yield of Grain Crops, CAB International, Wallingford, 178 pp. Egli, D.B., 1999. Variation in leaf starch and sink limitations during seed ®lling in soybean. Crop Sci. 39, 1361±1368. Egli, D.B., Zhen-wen, Y., 1991. Crop Growth rate and seed number per unit area in soybean. Crop Sci. 31, 439±442. Fehr, W.R., Caviness, C.E., 1977. Stages of soybean development. Spec. Rep. 80 Coop. Ext. Service. Iowa State University, Ames, IA. Fischer, R.A., 1985. Number of kernels in wheat crops and the in¯uence of solar radiation and temperature. J. Agric. Sci. 105, 447±461. Goldschmidt, E.E., Huber, S.C., 1992. Regulation of photosynthesis by end-product accumulation in leaves of plants storing starch, sucrose, and hexose sugars. Plant Physiol. 99, 1443± 1448. Heberer, J.A., Below, F.A., Hageman, R.H., 1985. Drying method affect on leaf chemical constituents of four crop species. Crop Sci. 25, 1117±1119. Huber, S.C., Rogers, H., Mowry, F.L., 1984. Effects of water stress on photosynthesis and carbon partitioning in soybean plants grown in the ®eld at different CO2 levels. Plant Physiol. 76, 244±249. Jiang, H., Egli, D.B., 1993. Shade induced changes in ¯ower and pod number and ¯ower and fruit abscission in soybean. Agron. J. 85, 221±224. Jones, P., Allen Jr., L.H., Jones, J.W., Boote, K.J., Campbell, W.J., 1984. Soybean canopy growth, photosynthesis and transpiration responses to whole-season carbon dioxide enrichment. Agron. J. 76, 633±637. Kiniry, J.R., Knievel, D.P., 1995. Response of maize seed number to solar radiation intercepted soon after anthesis. Agron. J. 87, 228±234. MacKown, C.T., VanSanford, D.A., 1986. Postanthesis nitrate assimilation in winter wheat. Plant Physiol. 81, 17±20. Miceli, F., Crafts-Brandner, S.J., Egli, D.B., 1995. Physical restriction of pod growth alters development of soybean plants. Crop Sci. 35, 1080±1085.
Mondal, M.H., Brun, W.A., Brenner, M.L., 1978. Effects of sink removal on photosynthesis and senescence in leaves of soybean (Glycine max L.) plants. Plant Physiol. 61, 394±397. Norman, J.M., Arkebauer, T.J., 1991. Predicting canopy light-use ef®ciency characteristics. In: Hanks, J., Ritchie, J.T. (Eds.), Modeling Plant and Soil Systems, ASA-CSSA-SSSA, Madison, WI, pp. 125±143. Peet, M.M., Kramer, P.J., 1980. Effects of decreasing source/sink ratio in soybeans on photosynthesis, photorespiration, transpiration and yield. Plant Cell Environ. 3, 201±206. Preiss, J., Sivak, M.N., 1996. Starch synthesis in sources and sinks. In: Zamski, E., Schaffer, A.A. (Eds.), Photoassimilate Distribution in Plants and Crops. Source±sink Relationships, Marcel Dekker, New York, pp. 63±96 Ramseur, E.L., Wallace, S.U., Quisenberry, V.L., 1985. Growth of `Braxton' soybeans as in¯uenced by irrigation and intrarow spacing. Agron. J. 77, 163±168. Schou, J.B., Jeffers, D.L., Streeter, J.G., 1978. Effects of re¯ectors, black boards, or shades applied at different stages of plant development on yield of soybeans. Crop Sci. 18, 29±34. Setter, T.L., Brun, W.A., 1980. Stomatal closure and photosynthetic inhibition in soybean leaves by petiole girdling and pod removal. Plant Physiol. 65, 884±887. Shibles, R.M., Secor, J., Ford, D.M., 1987. Carbon assimilation and metabolism. In: Wilcox, J.R. (Ed.), Soybeans: Improvement, Production and Uses, ASA-CSSA-SSSA, Madison, WI, pp. 535±588. Stephenson, A.G., 1981. Flower and fruit abortion: Proximate causes and ultimate functions. Annu. Rev. Ecol. Syst. 12, 253± 279. Thorne, J.H., Koller, H.R., 1974. In¯uence of assimilate demand on photosynthesis, diffusive resistance, translocation, and carbohydrate levels of soybean leaves. Plant Physiol. 54, 201±207. Vega, C.R., Andrade, F.H., Sadras, V., Uhart, S.A., Valentinuz, O.R., 1998. Grain yield and kernel number in maize, sun¯ower, and soybean as a function of growth. In: Otegui, M.E., Slafer, G.A. (Eds.), International Workshop on Physiological Basis for Maize Improvement, Buenos Aires, pp. 108±109.
Leaf starch accumulation and seed set at phloem-isolated nodes in soybean W.P. Bruening*, D.B. Egli Department of Agronomy, University of Kentucky, Lexington, KY 40546-0091, USA Received 20 April 2000; received in revised form 8 July 2000; accepted 11 July 2000
Abstract There is usually a linear relationship between pod and seed number per unit area and assimilate supply in soybean (Glycine max L. Merrill) communities. In contrast, the relationship is curvilinear at single phloem-isolated nodes. To further investigate this curvilinear relationship, we evaluated photosynthesis, leaf starch levels and pod and seed number at phloem-isolated nodes of cultivar Elgin 87 in three greenhouse experiments. The main stem was girdled between the ®fth and sixth nodes (uni®oliolate node was node one) when the ®rst ¯owers opened at the sixth node. The main stem above the sixth node was removed and defoliation (®ve or six levels from 0 to 100%) created a range in assimilate supply. Girdling increased leaf starch levels 3- to 7-fold over non-girdled plants within 7 days. In two experiments starch decreased to control levels within 14±27 days after girdling, but in a third experiment the increase was maintained for 28 days. Defoliation reduced leaf starch levels, with 66±91% defoliation lowering it to the level in the non-girdled controls. Pod and seed number were directly related to assimilate supplies at low levels of assimilate availability when there was no accumulation of starch. There were only relatively small increases in pod and seed number at high levels of assimilate availability, but there were large accumulations of starch in the leaves. Flower and pod abortion was always above 50%, so pod set was not limited by ¯ower availability. The failure of pod and seed number to respond to high levels of assimilate availability suggests that there may be other processes involved in determining pod and seed number at isolated nodes in soybean. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Leaf starch accumulation; Phloem-isolated nodes; Soybean; Defoliation; Girdling
1. Introduction The number of seeds per unit area is usually related to canopy photosynthesis during some critical stage of reproductive growth in most grain crops. For example, modi®cation of canopy photosynthesis or crop growth rate of soybean by shade (Egli and Zhen-wen, 1991; Egli, 1993), CO2 enrichment (Jones et al., 1984) or increased irradiance (Schou et al., 1978) caused cor*
Corresponding author. Tel.: 1-859-257-9404; fax: 1-859-257-7874. E-mail address: [email protected] (W.P. Bruening).
responding changes in pod and seed number. Similar responses have been reported for other crops (Fischer, 1985; Kiniry and Knievel, 1995). Varying the leaf area at a single phloem-isolated soybean node, created by girdling the main stem, demonstrated that increasing total photosynthesis or nodal carbon input (NCI) above a minimum level did not substantially increase pod or seed number in spite of 30±70% reproductive abortion (Bruening and Egli, 1999). Maximum pod and seed number frequently occurred at less than 50% of maximum NCI in two ®eld experiments with three cultivars. Similar curvilinear relationships were reported for individual plants of
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maize (Zea mays L.) and sun¯ower (Helianthus annuus L.) (Edmeades and Daynard, 1979; Vega et al., 1998). This curvilinear response at isolated nodes in soybean contrasts with the relationship at the community level, where seed number is usually linearly related to crop growth rate (an estimate of canopy photosynthesis) during ¯owering and podset (Ramseur et al., 1985; Charles-Edwards et al., 1986; Egli, 1993). The physiological basis for this difference between phloem-isolated nodes and plant communities is not known. Some of the carbon ®xed in photosynthesis may be converted to starch in the leaf and not immediately transported to developing sinks (Preiss and Sivak, 1996) suggesting that simple estimates of photosynthesis may not adequately describe the assimilate supply to developing reproductive structures. The amount of starch in a soybean leaf responds quickly to changes in source±sink ratios. For example, reducing sink size increased leaf starch content and decreased carbon exchange rate (CER) (Mondal et al., 1978; Miceli et al., 1995). Alternatively, decreasing the source±sink ratio by shading all but a few leaves decreased leaf starch content and/or increased CER of unshaded leaves for at least 8 (Thorne and Koller, 1974) or 22 (Peet and Kramer, 1980) days after treatment. Petiole girdling to block phloem export in soybean dramatically increased leaf starch content and reduced leaf CER for at least 7 days (Goldschmidt and Huber, 1992), however, over shorter intervals (between 0.5 and 12 h) leaf CER decreased, but there was no signi®cant effect on leaf starch content (Setter and Brun, 1980). The availability of assimilate to the reproductive sink is related to CER in the leaf and changes in leaf carbohydrate levels. Evaluation of changes in leaf starch and soluble carbohydrates at phloem-isolated nodes in soybean will contribute to a better understanding of the relationship between photosynthesis and pod and seed number. Thus, the objective of this study was to evaluate the relationship between leaf photosynthesis, starch content and pod and seed number at phloem-isolated nodes in soybean. 2. Materials and methods Three greenhouse experiments were conducted at the University of Kentucky during 1996 and 1998.
Seeds of soybean cultivar Elgin 87 (Maturity Group II) were planted in 3 l pots ®lled with a 2:1 (v/v) mixture of silt loam soil and vermiculite on 23 April 1996 (Exp. 1), 30 August 1996 (Exp. 2), and 16 July 1998 (Exp. 3). Pots were overseeded and thinned to one plant per pot after emergence. The plants were fertilized with 0.3 g per pot of Peters Professional fertilizer (20-20-20) at approximately weekly intervals. On 4 June 1996 (Exp. 1), 11 October 1996 (Exp. 2) and 18 August 1998 (Exp. 3) the sixth node (unifoliolate node was node one) on the main stem of each plant was isolated by girdling the stem below the node and removing the portion of the plant above the node. The stem was girdled when the ®rst ¯owers at the girdled node opened and the plants were at growth stage R1 (Fehr and Caviness, 1977). Girdling was accomplished by electrically heating a bare constantan wire (approximately 0.7 mm diameter) wrapped around the stem as described previously (Bruening and Egli, 1999). The girdle was considered a success if, after 2 days, a complete band of dead tissue was observed around the stem. Variation in assimilate production at the girdled nodes was created by removing 0 (non-defoliated), approximately 34 (middle lea¯et removed), 66 (two side lea¯ets removed), 83 (two side lea¯ets and half of middle lea¯et removed), 91 [two side lea¯ets and 75% of the middle lea¯et removed (Exp. 3 only)], and 100% (entire leaf removed) of the leaf area when the plants were girdled. To reduce assimilate supply by shading, plants from the non-defoliated treatment in Exp. 3 were placed under 63% shade cloth when they were girdled. Leaf area of the center and side lea¯ets removed from plants receiving the 100% defoliation treatment was determined to estimate the leaf area removed in the other defoliation treatments (Bruening and Egli, 1999). A control±cut treatment (non-girdled, non-defoliated plant with the main stem above the sixth node removed) was included to provide a basis of comparison of the effect of girdling on photosynthesis and leaf starch content. The plant above the girdled node was removed on all treatments, thus they were all equally exposed to any possible effect of removing the stem apex. A completely randomized experiment design was used with 6±15 replications (plants) per treatment. Girdling was not always successful resulting in a variable number of plants per treatment.
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After girdling and defoliation, leaf CER was measured in Exp. 2 and 3 with a LI-6400 portable photosynthesis system (Li-Cor, Inc., Lincoln, NE). The CER of four leaves per treatment was determined before girdling and at approximately 5-day intervals on relatively sunny days when photosynthetically active radiation (PAR) was generally greater than 1000 mmols mÿ2 sÿ1 (Exp. 2) between 1100 and 1400 h. In Exp. 3, leaves were exposed to a constant PAR of 1500 mmols mÿ2 sÿ1 during the CER determination with a Li-Cor 6400-02B LED Red/Blue Light Source. The total daily photosynthesis of the leaf determines the daily carbon input (DCI) at the girdled node. Photosynthesis in a well watered greenhouse environment should be determined primarily by PAR, air temperature and the inherent photosynthetic capacity of the leaf. Thus, daily CER could be estimated with a model that accounts for these variables. The PAR and temperature in the greenhouse were measured at the top of the plants and averaged over 0.5 h intervals with a Li-Cor 1000 data logger. A PAR response curve, created by measuring CER at 13 PAR levels from 50 to 2000 mmol mÿ2 sÿ1 on two replications of leaves from Elgin 87 plants (control±cut) in the greenhouse, was used to estimate CER from measured PAR levels. Regression analysis of the relative CER (where, Y CER expressed as a percent of CER at 1500 mmol mÿ2 sÿ1 PAR) resulted in Eq. (1) (R2 0:99, regression was signi®cant at P 0:0001). Y ÿ107:7087 28:4266 ln PAR
(1)
The CER for each of the six defoliation treatments for each 0.5 h period during the day was estimated by ®rst calculating Y from the measured PAR (Eq. (1)) and then converting it to CER by multiplying Y by the CER measured frequently at 1500 mmol mÿ2 sÿ1 (CER1500) for each treatment. Daily values of CER1500 were estimated by linear interpolation between the periodic estimates (four over the 14day period). Bruening and Egli (1999) demonstrated that defoliation of leaves at phloem isolated nodes affected CER, making these adjustments for treatment effects necessary. The estimated CER was adjusted for temperature using a CER-temperature response curve from Norman and Arkebauer (1991), however, day time greenhouse temperatures were usually in the range (26±368C) where their curve predicts little
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response to temperature. Average maximum PAR over the 14-day period was 1256 mmol mÿ2 sÿ1 with a range from 774 to 1423 mmol mÿ2 sÿ1. Total daily CER was multiplied by leaf area per node to get DCI per node. The root mean square error comparing predicted CER with CER determined at a range of ambient PAR levels was 2.1 mmols CO2 mÿ2 sÿ1 (n 13), thus, the estimated DCI provided an adequate representation of the effect of daily and diurnal variation in PAR and temperature on total CER. Lea¯ets at the girdled node were harvested between 1100 and 1400 h at approximately 7- (Exp. 1, 3 replications) or 5-day intervals (Exp. 2 and 3, 4 replications) for carbohydrate analysis. Leaves were placed on ice in the greenhouse, taken immediately to the laboratory where the leaf area was determined before they were frozen, freeze dried and ground for analysis after the dry weight was determined. Total extractable sugar and starch contents were determined using the techniques of Heberer et al. (1985). At maturity, the number of pods, seeds, and ¯ower scars (Jiang and Egli, 1993) at the girdled node was determined. The combined ¯ower and pod abortion was calculated from the number of ¯owers and mature pods. 3. Results and discussion There was a curvilinear relationship between DCI at the girdled node and pod and seed number in Exp. 3 (Fig. 1). Pod and seed number at the girdled node reached near maximum levels at relatively low DCI levels (<4 mmol CO2 per node per day). Similar curvilinear relationships were found in Exps. 1 and 2 and in ®eld experiments with this cultivar and two other cultivars (Bruening and Egli, 1999). Combined ¯ower and pod abortion was substantial at the high DCI levels (i.e. low levels of defoliation) in these greenhouse experiments (51±74%) (Table 1) and in previous ®eld experiments (32±71%) (Bruening and Egli, 1999), suggesting that the curvilinear response did not result from a lack of ¯owers. Defoliation had no signi®cant effect on the total number of ¯owers at the girdled node (data not shown). Defoliation had almost no signi®cant effect on seeds per pod (Table 1), so the variation in seeds per node was primarily determined by the fate of the developing pod. In
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generally accepted concept that the number of reproductive structures is directly related to assimilate supply (Stephenson, 1981; Charles-Edwards et al., 1986; Egli, 1998). Since the supply of assimilate to a developing reproductive structure is not necessarily equivalent to leaf CER, we decided to investigate the relationship between soluble sugar and starch levels in the leaf, and pod and seed set at phloem-isolated nodes. Starch levels in leaves at phloem-isolated nodes in all three experiments increased rapidly and signi®cantly (P 0:05) after girdling, reaching maximum levels within 6±7 days (Fig. 2). There was no change in starch levels in leaves from the non-girdled control plants in Exp. 1 and 2, but in Exp. 3 starch increased and there was no signi®cant difference between leaves from the control and girdled plants until the fourth sample (13 days after girdling). The difference between the control and girdled plants decreased with time in Exps 1 and 3, but in Exp. 2 girdled plants maintained high starch levels for nearly 28 days. Leaf starch levels in soybean respond quickly to alterations in source±sink ratios (Shibles et al., 1987; Egli, 1999), so the increase in leaf starch reported here probably re¯ects a sink limitation imposed by girdling. Similar starch accumulations after girdling have been reported for soybean (Goldschmidt and Huber, 1992), wheat (Triticum aestivum L.) (MacKown and VanSanford, 1986), pea (Pisum sativum L.) and cotton (Gossypium hirsutum L.) (Goldschmidt and Huber, 1992). Defoliation at the girdled node reduced total photosynthesis per node (Bruening, 1997; Bruening and
Fig. 1. The relationship between assimilate supply and pod and seed number at phloem-isolated nodes in soybean, cultivar Elgin 87, Exp. 3. The daily carbon input was averaged over the ®rst 14 days after the plants were girdled. The symbols , identify the 63% shade treatment. The regression analysis with pod and seed number was signi®cant at P 0:01. The bars represent standard error of the mean.
contrast, there was variation in seeds per pod among experiments with the plants in Exp. 3 producing, on the average, 44% more than plants in Exp. 1 and 2 (Table 1). The basis for this differential response is not known. As reported previously (Bruening and Egli, 1999), the failure of pod and seed number to increase rapidly at high DCI levels is obviously not consistent with the
Table 1 Effect of defoliation at phloem isolated nodes on seeds per pod and reproductive abortion in three greenhouse experiments, cultivar Elgin 87 Experiment
Defoliation (%) 0
Seeds per pod 1 2 3a
1.7 1.7 2.4
Flower and pod abortion (%) 1 58 2 74 3b 51 a b
LSD (0.05) 33 1.8 1.8 2.8 61 73 61
66 1.8 2.1 2.7 61 70 72
The 63% shade-non-defoliated treatment had 2.7 seeds per pod. The 63% shade-non-defoliated treatment had 55% abortion.
83 1.8 1.8 2.6 69 72 72
91 ± ± 2.5 ± ± 80
NS NS 0.3 10 NS 10
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Fig. 2. The effect of girdling the main stem to block phloem transport on starch levels (g mÿ2 leaf area) in the leaf at the girdled node in three greenhouse experiments. The bars represent the LSD (P 0:05) for comparison of treatments at any sampling date.
Fig. 3. The effect of defoliation and shade on starch levels (g mÿ2 leaf area) in leaves at the girdled node in three greenhouse experiments. The bars represent the LSD (P 0:05) for comparison of treatments at any sampling date.
Egli, 1999) and signi®cantly (P 0:05) reduced the accumulation of leaf starch in all three experiments (Fig. 3). These reductions were generally in proportion to the degree of defoliation, except in Exp. 3 where there were no consistent differences among the 66, 83, and 91% defoliation treatments. Shading (63%) nondefoliated plants in Exp. 3 reduced starch to levels found in the highest defoliation treatments. Starch levels at the highest levels of defoliation in all experiments were generally similar to or below levels in ungirdled control plants (Figs. 2 and 3). Reducing photosynthate production per node by defoliation apparently reduced the source±sink imbalance created by girdling and reduced starch accumulation in the
leaves, a common response to reduced source activity (Huber et al., 1984; Shibles et al., 1987; Egli, 1999). Rather large reductions in DCI (66% defoliation) were required to reduce starch levels to those found in non-girdled plants, suggesting that approximately half of the assimilate was translocated away from the node subtending the sixth leaf (i.e. the node that was girdled) in control plants. Soluble sugars were not in¯uenced by defoliation in Exp. 2 and 3, but in Exp. 1 the soluble sugar levels followed the changes in starch (Fig. 4). There was no signi®cant difference in soluble sugar levels between leaves on control (non-girdled) and non-defoliated girdled plants, except for the 7-day sample in Exp. 1
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the response curve de®ned by the defoliation treatments (Fig. 1). Excessive accumulation of starch is an indicator of a sink limited system, i.e. the supply of assimilate exceeds the capacity of the sinks (Shibles et al., 1987). Leaf starch accumulated at high DCI levels (e.g., defoliation 66%, Fig. 3), but in spite of this apparent excess assimilate supply, there were only small increases in pod and seed number. The relatively large reductions in pod and seed number as the DCI decreased below 4 mmol CO2 per node per day (defoliation > 66%) in Exp. 3 (Fig. 1) were associated with minimal changes in average leaf starch levels (4.4±5.4 g mÿ2, Fig. 5). However, the relatively small changes in pod and seed number at higher DCI levels in all three experiments were associated with large increases in average leaf starch, starting at approximately 5 g mÿ2. This bi-phasic relationship between
Fig. 4. The effect of defoliation and shade on soluble sugar levels (g mÿ2 leaf area) in leaves at the girdled node in three greenhouse experiments. The bars represent the LSD (P 0:05) for comparison of treatments at any sampling date.
(data not shown). Thus, girdling and defoliation affected starch levels without consistently affecting soluble sugars. In contrast, shading non-defoliated plants reduced both starch and sugar levels in Exp. 3, although the effect on starch was much larger that the effect on soluble sugars (Figs. 3 and 4). Apparently altering source±sink ratios by reducing photosynthesis per unit area with shade elicited a different response in soluble sugar levels than reducing total photosynthesis per node by defoliation. This differential response did not seem to in¯uence the relationship between DCI and pod and seed number. The data from the shade treatment are consistent with
Fig. 5. The relationship between average leaf starch levels (g mÿ2 leaf area) for all samples from 1 to 14 days after girdling and pods and seed per node on girdled plants in three greenhouse experiments.
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starch and pod and seed number illustrates the apparent change in sink limitations that occurred as assimilate availability increased. Below approximately 66% defoliation, pod and seed number were closely associated with assimilate availability and there were minimal changes in leaf starch levels. The starch level in these leaves (5 g mÿ2) was similar to those found in non-girdled plants, suggesting that non-girdled plants may also exhibit a close association between pod and seed number and assimilate supplies. This biphasic relationship was not evident in Exps. 1 and 2 because the defoliation treatments did not reduce DCI and leaf starch to low enough levels (Fig. 5). Apparently when the assimilate supply increased to a critical level, pod set stopped and the leaves began to accumulate starch, re¯ecting a sink limitation. This part of the bi-phasic response, which is dif®cult to understand given the high level of ¯ower and pod abortion, may be related to the timing of ¯owering and pod development. The ®rst ¯owers and pods to develop may not initially provide a large enough sink to use all of the assimilate, resulting in starch accumulation. However, the assimilate available to ¯owers and pods developing later may be limited when the seeds in these early pods enter the rapid growth phase and consume relatively large amounts of assimilate. This lack of assimilate may cause late developing ¯owers and pods to abort, as we proposed earlier (Bruening and Egli, 1999). The eventual decline in leaf starch levels in Exp. 1 and 3 (Fig. 2) is consistent with this scenario. However, such declines in starch did not occur in Exp. 2, raising the possibility that late pod development may have also been prevented by a dominance type mechanism, possibly involving hormones (Bangerth, 1989). Clearly, much remains to be learned about the mechanisms regulating pod and seed set in soybean. The relationship between average leaf starch levels and pods per node was similar across experiments (Fig. 5), but there were large differences among experiments in seed number per node. The plants in Exp. 3 produced more seeds per node and per pod (Table 1) than the plants in Exp. 1 at the same starch level. The reason for these differences is not clear, but the results emphasize that both pod and seed set are involved in the determination of seed number. Pod number is determined by the number of ¯owers and the proportion that produce mature pods. Individual
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seeds in a pod may also develop or abort and seed number is the end result of these two processes. Abortion of pods probably occurs ®rst with seed abortion occurring only after the pod is fully developed. The variation in Fig. 5 may re¯ect an interaction between the greenhouse environment and this difference in occurrence of the critical growth stage. Seed number was also more responsive to higher DCI levels than pod numbers in Exp. 3 (Figs. 1 and 5), further evidence of a possible environmental interaction. In summary, pod and seed number at phloem-isolated nodes was only closely related to assimilate supplies at relatively low levels of assimilate availability. Pod and seed number exhibited only small increases at high levels of assimilate availability, but there were large accumulations of starch in the leaves, indicating that pod and seed number was not limited by assimilate supply. The failure of pod and seed number to respond to the greater assimilate availability suggests that there may be other processes, in addition to assimilate supply, involved in determining pod and seed number at isolated nodes in soybean. The exact nature of these processes remains to be determined. Acknowledgements Paper no. 00-06-62. Published with the approval of the Director of the Kentucky Agricultural Experiment Station, University of Kentucky, Lexington. References Bangerth, F., 1989. Dominance among fruits/sinks and the search for a correlative signal. Physiol. Plant 76, 608±614. Bruening, W.P., 1997. The relationship between photosynthesis and seed number in soybean cultivars with differences in seed size. M.S. Thesis, University of Kentucky, Lexington, KY. Bruening, W.P., Egli, D.B., 1999. Relationship between photosynthesis and seed number at phloem isolated nodes in soybean. Crop Sci. 39, 1769±1775. Charles-Edwards, D.A., Doley, D., Rimmington, G.M., 1986. Modeling Plant Growth and Development. Academic Press, Sydney, Australia, 235 pp. Edmeades, G.O., Daynard, T.B., 1979. The relationship between ®nal yield and photosynthesis at ¯owering in individual maize plants. Canad. J. Plant Sci. 89, 585±601. Egli, D.B., 1993. Cultivar maturity and potential yield of soybean. Field Crops Res. 32, 147±158.
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