Pot experiment suggests that the low yields of a super-nodulating soybean (Glycine max L. En-b0-1)
was not caused by N
2fi xation activity of nodules itself.
Hideo Hamaguchi
a*, Akinori Takeda
a**, Toshio Sugimoto
a,b,†, Tetsushi Azuma
aaFaculty of Agriculture, Graduate School of Agricultural Science, Kobe University (Rokkodai 1, Nada, Kobe, Hyogo, Japan) bResearch Center for Food and Agriculture, Wakayama University (Sakaedani 930, Wakayama, Wakayama, Japan)
*Western Region Agricultural Research Center, NARO (Fukuyama, Japan) **Suita City Hall (1-3-40 Izumi, Suita, Osaka, Japan)
Summary: Although super-nodulating (SN) cultivars (cvs) of soybean had been developed for the improvement
of seed yields of soybean, seed yields of all SN cvs developed until now were much less than those of normally nodulated soybean plants. The mechanism why plants of SN cv yielded less seed than those of normal nodulating cv was not clear yet. We compared the plant growth and seed productions from three genotypes of a near isogenic line (NIL) of soybean plants having different nodulation activities, i.e., Enrei (a popular cv in Japan), En-b0-1 (a SN genotype produced from Enrei) and En1282 (a non-nodulating genotype produced from Enrei). We grew plants of them on soils in pot having low- and high-N levels to assess the eff ects of nodulation and volume of soil where plants can develop their roots since soil N inhibit the nodulation and N2 fixation
activities of soybean plants. We observed that plants from all the three genotypes showed almost the same seed weights per plant with each other except that of En1282 in low-N soil. Our potted experiment showed that higher N2 fi xation activity of En-b0-1 plants did not cause the decrease in seed weight per plant. Based on these
results, it was suggested that the N2 fi xation activity of nodules was not a cause of poor seed productions in the
SN genotype of soybean, En-b0-1, in fi elds.
Keywords: near isogenic line, seed yield, urea coated slow-release nitrogen fertilizer, soybean,
super-nodulation
1. Introduction
Soybean plants are able to fix atmospheric nitrogen (N2) in
nodules on their roots, and they use the fi xed N for plant growth. Soybean super-nodulating (SN) plants were developed (Carroll
et al. 1985) with the goal of improving the yields of seeds by
satisfying the high N demand of the seeds without the use of N fertilizer. Contrary to the expectations, however, the yields of the soybean SN plants were less than those of normal (mid-nodulating, MN) soybean plants (Wu and Harper 1991, Akao and Kouchi, 1992, Pracht et al 1994). Those results suggested that an excess consumption of photosynthate by the soybean plants nodules caused the low seed yields.
MN plants use N derived from both symbiotic N2 fi xation and
the assimilation of mineral N in the soil (including fertilized N), both the N2 fi xation and the N assimilation activities of the plants
were important for the maximum seed yield (Ohyama et al. 2013). In SN plants, the nodules on the roots were thought to supply a quantity of N to maturing seeds that was greater than the seeds demand for N. Non-nodulating (NN) soybean plants
have no nodules and no N2 fixation activity, and it thus
appropriate to use NN plants as a control for evaluations of the eff ects of nodulation on seed yields.
A 1985 study by Streeter showed that the soil s N content can inhibit both nodule formation and the N2 fi xation activity of the
nodules. Urea coated slow-release nitrogen fertilizers (SNFs) secrete N on a constant rate within certain periods depending on their characteristics (JCAM Agri. 2018), which affects the N2
fi xation activities and the accumulation of storage compounds in developing soybean seeds (Sugimoto et al. 2001).
We conducted the present study to clarify why poor yields are observed in SN soybean plants by comparing the growth and seed productions of plants grown in the potted condition of three genotypes of soybean, a cv Enrei and its two near-isogenic lines (NILs) having diff erent N2 fi xation activities.
2. Materials and methods
Fertilizers, plant materials, chemicals, instruments and designation of growth conditions.
We used a compound fertilizer (N:P:K = 8:8:8, Shoken Sangyo, Chiba, Japan), and one type of SLNF (MEISTER-15, Acccepted: July 22, 2019
Corresponding author: †Toshio Sugimoto ([email protected])
M-15) having a 100-day lifespan, a product of JCAM Agri (Tokyo, Japan). The SLNF secrete N on a constant rate through plant growth as shown in http://www.jcam-agri.co.jp/en/product/ meister.html.
The soil used was the plant culture soil Yanmer-S (Yanmer Co., Osaka, Japan), which provides its N supply to seedlings within 10 days after germination (DAG). The polyethylene pots used was 22.5 cm high, 10.5 cm in diameter at the top, and 7.5 cm diameter at the bottom. Zeolite was a product of Soft Silica Co Ltd, Tokyo (Million™).
Soybean (Glycine max. L.) seeds used in this experiment were as follows: (1) The MN cs Enrei, which is commonly used in Japan (JP No. 28862 in the Ministry of Agriculture, Forestry and Fisheries [MAFF] Gene Bank, Japan (https://www/gene.affrc. go.jp/database-plant_search_en.php) which has normal and moderate nodulation ability. (2) En-b0-1, a SN mutant of Enrei, which is a hybrid between Enrei and a SN plant, En6400 (Akao and Kouchi 1992) that is produced by the ethyl methane sulphonate (EMS) treatment of Enrei seeds (Takahashi et al. 2003). (3) En1282, a NN mutant of Enrei produced by EMS treatment (Francisco and Akao 1993).
The chemicals used in this experiment were guaranteed reagents from Nacalai Tesque (Kyoto, Japan). The insecticide used, Trebon® (etofenprox) was a product of Mitsui Chemicals
Agro (Tokyo).
A chlorophyll meter (SPAD-502, Konica Minolta, Tokyo, Japan) was used for the measurement of soil and plant analyzer development (SPAD) values of leaves.
Soils fertilized with the SLNF were designated as high-N soil (H-N) and soils without the SLNF were as low-N soil (L-N), respectively. Pots were set on the trolley on concrete fl oor in the open air at Kobe University (34 °43 27 N, 135 °13 58 E) in 2015 summer.
Plant growth condition
The plant culture soil was mixed thoroughly with approx. 0.021 g/l NH4NO3, 0.037 g/l KH2PO4, 0.086 g/l MgSO47H2O and
57 g/l zeolite. For the H-N, 0.5 g/l NaNO3 and 0.9 g/l of the
SLNF, M-15, were added to the soil. Two seeds were placed in each pot on June 16, 2015 and germinated. The pots were placed on trays from which water was supplied from bottom holes of pots. Our measurements of the plant height and the SPAD values of the leaves, and the collection of xylem saps were carried out during plant growth. Pods were collected after 94 days after germination and counted their number per plant. We appropriately applied the insecticide Trebon during the maturation stage of the seeds to eliminate the damage of developing seeds by bugs. The numbers of plants examined are listed in Table 1.
Estimations of N2 fixation activities and measurements of
plant growth, and seed production
The method to collect xylem saps was as described (Masuda et al. 2003). The collected xylem saps were diluted by 4 vol. of 100% ethanol and stored in a refrigerator ( − 20°C) until use. The concentrations of nitrate, amino acids, and ureides in xylem saps were spectrophotometrically measured by the methods of Cataldo et al. (1975), Moore and Stein (1954), and Young and Conway (1942), respectively. Ratio of N derived from N2
fixation of plants (Ndfa, %) was estimated by measuring concentrations of the three nitrogenous compounds, ureide, amino acids and nitrate using the equation proposed by Takahashi et al (1992). The equation was that Ndfa (%) = 4 x (ureide) / {4 x (ureide) + 4 x (amino acids) + (nitrate)} x 100.
The SPAD values of leaves reported herein are the averages of those from 10 leaves of a plant. The plant heights were measured manually, and the numbers of pods and seeds per plant were counted manually. Seed weight per plant was measured at > 3 months post-harvest.
Statistical analysis
Data were analyzed by the Tukey s honest signifi cant diff erence test and Student t-test as described in the legend of Table 2.
3. Results
The eff ects of N fertilization on the plant growth of the three genotypes
Figure 1 illustrates the changes in plant heights and SPAD values of plants of the three genotypes during their growth. The height of the plants of the three genotypes grown in the H-N in pots was almost the same as those of the corresponding plants grown in L-N, even though slight diff erences were observed in each genotype. After 30 DAG, the SPAD values of the leaves of the En1282 plants grown in pots of L-N were much lower than those of the En1282 plants grown in H-N, whereas the SPAD values of the Enrei and En-b0-1 plants were almost the same between the plants grown in H-N and L-N, respectively.
Table 2 lists the seed weight per plant, seed weight per seed, seed number per plant, and pod number per plant of the plants of the three genotypes. For the En1282 plants, all of these parameters values were higher for the plants grown in H-N compared to those of the plants grown in L-N, whereas for the other two genotypes there were much less diff erences in these parameters between the plants grown in the two N-level soils.
The values of these parameters were not signifi cantly diff erent between Enrei and En-b0-1 plants grown in the same types of soil except those of the seed weight per seed in L-N. Those values of Enrei and En-b0-1 plants were signifi cantly higher than that of En1282 plants grown in L-N.
The effects of N fertilization on the N metabolisms of the three genotypes’ plants
The concentrations of nitrogenous compounds, ureide, amino acids, and nitrate in xylem saps from the plants grown in pots were greatly changed, depending on characteristics of the plants during plant growth (Figure 2). At 36 DAG, high concentrations of ureide were detected in Enrei and En-b0-1 plants grown in L-N, and low concentrations of ureide were also detected in xylem saps from almost all of the Enrei, En-b0-1 and En1282 plants grown in H-N. The ureide concentrations in xylem saps from the Enrei and En-b0-1 plants grown in L-N at 36 and 41 DAG were high. On 19 DAG, the amino acid concentrations of the plants of all three genotypes grown in H-N were high. The concentrations of nitrate from the plants grown in H-N were higher than those of the plants grown in L-N at 19 DAG, but these concentrations decreased to almost the same level as those of the plants grown in L-N after 30 DAG in all three genotypes. The concentrations of nitrate of all three genotypes in both the
L-N and H-N (except for the En-b0-1 plants grown in L-N) were gradually decreased during the plant growth. Changes in the ratios of N derived from N2 fi xation in total N in xylem saps was
shown in the inserted table in Figure 2. The Ndfa of plants grown on H-N soil at 30 to 41 DAGs were lower than those grown on L-N soil of both nodulating genotypes.
As NN soybean plants is also able to synthesize ureide (Takahashi et al. 1991) and the concentration of ureide and those of amino acids in xylem saps from En1282 plants were correlated with each other (Masuda et al, 2003), it is useful to compare the ratio of the concentrations of ureide and amino acids in xylem saps for more accurate assessments of the N2
fi xation activity and N assimilation of plants. We compared the concentrations of the two nitrogenous compounds, ureides and amino acids, in xylem saps from plants of nodulating genotype with those of NN genotype (Figure 3) for quantitatively assessing the supression rate of N2 fi xation activities of plants of
nodulating genotypes by the N fertlization,. The coeffi cient line Table 1. Numbers of plants used for the measurements of the plant height and SPAD value and the seed yields of the plants
grown in pots
genotype Soil N DAG 20† 36‡ 44 65 94§
En1282 Low 40 23 18 18 18 High 39 23 18 18 15 Enrei Low 38 23 18 18 18 High 40 24 19 19 19 En-b0-1 Low 38 23 18 18 18 High 40 25 20 20 20
Abbreviation DAG：days after germination
† The DAG of measuring SPAD value of all plants was 19.
‡ The DAG of measuring plant height of plants grown on Low-N soil was 37.
§Seed weight / plant of all plants were measured after drying seeds for more than three months.
Table 2. Diff erences in the seed production among three soybean genotypes with diff erent nodulation abilities
Genotype Seed weight/plant, g Seed weight / seed, g Seed number / plant Pod number / plant Soil type Low-N High-N Low-N High-N Low-N High-N Low-N High-N En1282 1.01 b 5.05†a 0.149 c 0.194†a 6.8 b 26.3†a 10.7 b 41.1†b
Enrei 4.96 a 6.54†a 0.185 b 0.208 a 26.3 a 31.1 a 68.2 a 76.8†a
En-b0-1 5.90 a 5.99 a 0.216 a 0.181†a 28.6 a 33.9 a 73.5 a 79.7 a
† indicates signifi cant diff erence (P<0.05, Student's t-test) between the low and high N treatments of each genotype.
Diff erent letters within columns indicate signifi cant diff erences (P< 0.05, Tukey's honest signifi cant diff erence test) among three genotypes of the same N treatment.
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Figure 1. Eff ects of N fertilization on the growth of plants of the three genotypes (Pot experiment). Left panels: Changes in stem length during plant growth. Right panels: Changes in SPAD values of leaves during plant growth. A: En1282. B: Enrei. C: En-b0-1. Open and solid symbols indicate the low and high N conditions, respectively. Circles: En1282. Squares: Enrei. Triangles: En-b0-1. The numbers of samples measured are listed in Table 1.
Changes in the ratio (%) of fixed N in total N (Ndfa) in xylem saps from plants of two nodulating genotypes. N DAG 19 30/36* 41 Enrei L 25.7 90.7* 80.7 H 29.3 24.6 47.1 En-b0-1 L 52.2 94.0* 88.0 H 37.6 41.9 83.2 0 300 600 900 Ureide, P M in xylem sap L H
En1282 L HEnrei L HEn-b0-1
Amino acids P M in xylem sap L H
En1282 L HEnrei L HEn-b0-1
Nitrate P M in xylem sap L H
En1282 L HEnrei L HEn-b0-1
(A)
(B) (C)
Figure 2. Changes in the concentrations of nitrogenous compounds, (A) ureide, (B)amino acids, (C) nitrate, in xylem saps from plants of the three genotypes during the early stage of plant growth.
Open, soild and striped bars indicate the days of collecting xylem saps, from left to right, 19, 30 and 41 days after germination, respectively. Marks * indicate the collecting date as 36 instead of 30 in the case of plants grown in low N condition.
Figure 3. Relations in the concentrations of amino acids and ureide in xylem saps from plants of the three genotypes during plant growth.
Symbols indicate as follows. Open and solid symbols indicate the low and high N conditions of soil, respectively, and circles, squares and triangles indicate genotypes of plants, En1282, Enrei and En-b0-1, respectively. Numbers beside marks show the day after germination of the samples collected. Dotted line indicate the relationship between the concentrations of ureide and amino acids in xylem saps from En1282 plants. The equation of their relation is y = 0.1355x + 3.8717 (y: ureide, x: amino acids, r = 0.765, n=6).
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between concentrations of the two nitrogenous compounds in xylem saps of En1282 plants were used as a standard to compare changes in the two activities, N2 fi xation and N assimilation, of
the two nodulating genotypes in each growth conditions. It was estimated simultaneously both the ratio between N2 fi xation on
N metabolism in plants and their activities during the early stage of plant growth. We observed that the ureide and amino acids concentrations in xylem saps from plants of both the nodulating plants grown in L-N were high and low, respectively, at 36 DAGs; the N2 fi xation activities of these nodulated plants were
thus high at the middle stage of plant growth. Activities of the N assimilation and N2 fixation of plants grown in H-N of the
nodulating genotypes were high and low, respectively, because the plots of the obtained data (ureide and amino acids) were located near the dotted line in Figure 3.
4 Discussion
Poor seed productions had been observed in plants of SN cvs of soybean as described in Introduction. A most probable cause of the poor productivity of SN soybean plants were thought to be ascribed to too many nodules in roots because nodules required much sugar for the energy source of N2 fixation. Another
characteristic on the structure of SN soybean plants is poor root development as Hamaguchi et al reported that the root weights of plants of En-b0-1 was half of those of Enrei (2016). We intended to compare the plant growth and seed productivity of
plants of En-b0-1 with those of Enrei, a MN NIL of En-b0-1, under the condition where N2 fi xation activity of nodules were
suppressed.
Although the applied N fertilizer suppressed N2 fixation
activities of plants of both nodulating genotypes during the early growth stage of plants (Figs. 2 and 3), the En-b0-1 plants showed the similar plant growth and seed production with Enrei irrespective of rates of N2 fixation activities (Table 2). These
results implied that there was other factor(s) except N2 fi xation
activity causing low yields of SN plants in fi eld conditions. We also observed that the seed weights per plant of the three genotypes were almost the same irrespective of N fertilization conditions except the seed weights per plant of En1282 in L-N (Table 2). Seed weights per plant from plants of both nodulating genotypes in this potted experiment was far less than those grown on fields in agriculture. These results led an idea that insuffi cient root growth of En-b0-1 and Enrei plants limited seed weights per plant. Even though N2 fi xation requires much energy
(photosynthate), photosynthesis was reported to not limit the activity of nodules of a SN mutant of Medicago (Cabeza et al 2014). That leaf greenness is a good indicator of photosynthetic activity of soybean plants (Ma et al 1995) supported that all of plants of the two nodulating genotypes supplied sufficient amounts of photosynthate to roots including nodules in L-N in the present experiments. Therefore, the contrasting results on seed yields of En-b0-1 plants between our present result and the field experiment reported previously (Takahashi et al, 2003)
suggested that the N2 fi xation of plants did not directly aff ect the
seed yields in fi elds and also suggested that the N supply from nodules was not a limiting factor for the seed yields of the SN plants. An important difference in growth conditions between our potted experiments and fi eld experiments of others was the volume of soil in which the plants developed their roots. The volume of each pot (approx. 1.3 L/pot; 4.5-cm radius and 20-cm height) was far less than the volume of soil in field in which plants can develop their roots (approx. 28 L/plant when a 30-cm radius and 30-cm soil depth are assumed). As nodules consume photosynthate as a strong sink, the possibility that carbon supply was not enough for root growth of En-b0-1 plants is not eliminated even under the condition N2 fi xation activity of plants
from En-b0-1 is almost completely suppressed. Some mechanism to control root development of the En-b0-1 plants is supposed to work.
References
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ポット試験で，根粒超着生ダイズ（
Glycine max L. En-b0-1）の低収量の
原因は，窒素固定活性ではないと示唆された
濱口秀生
a*・武田旭永
a**・杉本敏男
a，b，†・東 哲司
a a 神戸大学大学院農学研究科 b 和歌山大学食農総合研究所 現在の所属 * ：農研機構西日本農業研究センター **：吹田市役所 要旨：ダイズの収量を増やす目的で育成された根粒着生数が非常に多い超着生ダイズ系統（SN）の種子収量は親の普通の根粒着 生数の親品種よりもかなり劣っている．この SN の普通品種に比べて低い収量の原因は不明であった．我々は，通常着生のエン レイとそれから育成された非着生系系統の En1282，超着生系統の En-b0-1 を土壌窒素濃度の異なる 2 つの条件，すなわち，根粒 着生が抑制される土壌窒素濃度の高い条件と根粒着生が促進される土壌窒素濃度の低い条件で根域を制限した小さなポットでの 栽培を行った．それらの登熟途中の導管液中の窒素化合物濃度を測定し窒素固定活性を推定するとともに完熟時の植物個体当た りの種子重量などを比較した．高濃度の土壌窒素は窒素固定活性を阻害したが，3 系統の植物個体当たりの種子重量は，低窒素 土壌での En1282 以外に差は見られず，En-b0-1 と Enrei では窒素固定活性の多寡は収量に影響しなかった．En-b0-1 の根粒着生量 の多さが低収量の原因ではないことが示唆された．キーワード：準同質遺伝子系統，種子収量，尿素コート緩効性窒素肥料，ダイズ，超着生
作物研究 65 号（2020） * 連絡責任者：杉本敏男（[email protected]）
