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a Legume Breeding Laboratory, National Agriculture Research Center, Kannondai, Tsukuba, Ibaraki, 305-8666 Japan
b Faculty of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan
masako{at}narc.affrc.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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15°C) at flowering induces browning around the hilum region and cracking of the seed coats. Both pigmentation and cracking degrade the external appearance of soybean seeds and reduce their commercial value. An earlier study showed that one of the genes responsible for pigmentation is closely associated with a maturity gene. The objective of this study was to evaluate the effect of five soybean maturity genes (E1–E5) on the intensity of seed coat pigmentation and cracking. Soybean cv. Harosoy (e1e2E3E4e5) and its near-isogenic lines (NIL) for E1 to E5 loci were exposed to 15°C for 2 wk beginning 8 d after anthesis. Control plants were grown in a greenhouse throughout their life cycle, whereas treated plants were transferred from the greenhouse to a phytotron for the chilling treatment. Intensity of pigmentation was not affected by e3, slightly reduced by E2 and e4, and profoundly reduced by E1 and E5. Degree of cracking was slightly increased by e3 and drastically reduced by e4, E1, and E5. The results suggest that some of the soybean maturity genes have inhibitory effects on the intensity of seed coat pigmentation and cracking in response to low temperatures. Dominant alleles E1 and E5 are most effective in suppressing both pigmentation and cracking. Therefore, these two genes may be useful to ensure tolerance to chilling stress in cultivars with e3 and e4, which jointly condition the insensitivity to long daylength, an adaptive trait in high latitude regions.
Abbreviations: DAA, days after anthesis of individual plants • DAO, days after opening of individual flowers • ILD, incandescent long daylength • NIL, near-isogenic line
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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of the temperate zone are sensitive to nonfreezing temperatures (10–18°C) as are plants from tropical and subtropical climates. Soybean cultivated at high latitudes and altitudes frequently suffers from low temperatures. Chilling stress usually retards growth, causes abortion of flowers and immature pods, and reduces the final seed yield (Raper and Kramer, 1987). Furthermore, chilling temperatures (15°C) during flowering induce browning and cracking of the seed coat (Sunada and Ito, 1982). In 1987, when mean temperature of the flowering period was 16.4°C, 39% of seeds produced in Hokkaido (northern Japan) developed a brown pigmentation around the hilum region in the seed coat (Hokkaido Department of Agriculture, 1988, unpublished data). In particular, `Toyomusume', the leading cultivar of Hokkaido, is quite susceptible to chilling temperatures. Pigmented seeds accounted for 59, 48, 85, and 21% of the yields at Tokachi Agric. Exp. Stn. in 1987, 1991, 1995, and 1997, respectively (Hokkaido Department of Agriculture, 1998). Brown pigmentation was also observed in Austria (Dr. J. Vollmann, 1996, personal communication) and in Croatia (Dr. V. Hrust, 1998, personal communication), where early-maturing soybeans are grown. The brown pigmentation looks similar to some of the symptoms caused by infection of soybean mosaic virus (Koshimizu and Iizuka, 1963), and it has been occasionally misclassified. The brown pigment induced by low temperature was probably formed by oxidation of phenolic compounds (Takahashi and Akiyama, 1993).
In Japan, cultivars with yellow hilum color are preferred to those with brown hilum for confectionery use due to better external appearance and higher protein content (Takahashi and Asanuma, 1996). However, yellow hilum cultivars are more susceptible to low temperatures, resulting in reduced seed yield and poor seed quality compared with brown hilum cultivars (Takahashi and Asanuma, 1996; Takahashi, 1997). Seed coat pigmentation occurs only in yellow hilum cultivars and is absent in cultivars with brown hilum (Sunada and Ito, 1982). Both pigmentation and cracking degrade the external appearance of soybean seeds and reduce their commercial value. Thus, chilling tolerance is one of the most important traits in cultivars adapted to high latitude regions, where chilling temperatures frequently prevail throughout the soybean growing season.
To clarify the genetic basis of genotypic differences in chilling tolerance. Takahashi and Asanuma (1996) and Takahashi (1997) evaluated the roles of gene T (responsible for pubescence and hilum color) and gene I (responsible for distribution of seed coat color) using NILs for the two loci. Independent of the genotypes at I locus, the dominant allele T completely suppressed the development of pigmentation around the hilum region and partly suppressed seed coat cracking. Dominant allele I also suppressed seed coat pigmentation and cracking under the genotype of tt, although its inhibitory effect was not as obvious as gene T. Genes T and I are involved in flavonoid biosynthesis. Gene T is assumed to encode a flavonoid 3'-hydroxylase, which hydroxylates the 3'-position of the B-ring in flavonoids (Buttery and Buzzell, 1973). Flavonoids with 3',4'-dihydroxy configuration possess a high antioxidant activity relative to those with a single hydroxyl group on the B-ring (Pratt, 1976). Most likely, the dominant allele T suppresses pigmentation by inhibiting the oxidation of phenolic compounds.
A multiple-allele locus I/i-i/i-k/i controls the distribution of the seed coat colors (Palmer and Kilen, 1987). I results in the complete absence of coloration over the entire seed coat, i-i restricts color to the hilum region, i-k limits color to the hilum and a saddle-shaped region surrounding it, and i results in a self-colored seed coat. The dominance relationships among the alleles of the I locus are: I > i-i > i-k > i. Dominant allele I encodes three tandem repeats of chalcone synthase, a key enzyme for flavonoid biosynthesis. It may inhibit the expression of chalcone synthase genes through a naturally occurring homology-dependent gene silencing, resulting in the complete absence of coloration (Todd and Vodkin, 1996). Seed coats of soybeans with recessive allelic combination of the two loci (i and t, or i-k and t) were severely cracked irrespective of environmental conditions (Nicholas et al., 1993). An increased level of chilling stress appears to surmount the function of allele I and consequently cause both pigmentation and cracking on the seed coat (Takahashi, 1997). Although both dominant alleles T and I suppress the pigmentation and cracking, the allelic combination of TI darkens the entire seed coat and is generally avoided in soybean breeding in Japan.
In addition to genes T and I, there is a genetic variation in the tolerance to pigmentation among cultivars with yellow hilum (I) and gray pubescence (t) (Takahashi and Abe, 1994). Analysis using F1 hybrids between chilling temperature sensitive and tolerant cultivars and their F2 population revealed that susceptibility was partially dominant to tolerance as a whole, and one or two major genes were involved in tolerance (Takahashi and Abe, 1994). However, a progeny test for the tolerant F2 plants suggested that at least one of the genes may be dominant and hypostatic to the others (Takahashi, 1992, unpublished data). Takahashi and Abe (1994) further found that one of the genes for the tolerance was closely associated with a dominant gene for late maturity, the recessive allele of which was involved in floral induction under artificially induced long days by means of incandescent lamps.
Six loci have been reported to control time to flowering and maturity in soybean: E1 and E2 (Bernard, 1971), E3 (Buzzell, 1971), E4 (Buzzell and Voldeng, 1980), E5 (McBlain and Bernard, 1987), and J for long juvenility (Ray et al., 1995). Of these six loci, E3 and E4 are known to be involved in the response of flowering to long daylength. The e3 locus controls the insensitivity to fluorescent long daylength obtained by extending natural daylength to 20 h using cool white fluorescent lamps with a low FR/R ratio, whereas e4 combines with e3 to control the insensitivity to incandescent long daylength (ILD) by extending natural daylength to 20 h using incandescent lamps with a high FR/R ratio (Buzzell, 1971; Buzzell and Voldeng, 1980). Another gene also was reported to be involved in ILD insensitivity (Abe et al., 1998). This study was conducted to further investigate the relationship between maturity genes and low temperature response in soybean, using NIL of Harosoy for the maturity genes.
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Materials and methods |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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Chilling treatment was carried out by transferring plants from the greenhouse to a phytotron set at 15 ± 0.5°C (day/night). Light was supplied at a photosynthetic photon flux density (400–700 nm) of 250 µmol m-2 s-1 using metal halide lamps (DR 400/T(L), Toshiba Co., Japan) at a 14 h light/10 h dark regime in the phytotron. Earlier studies revealed that pigmentation due to chilling is most intense when flowers from 5 to 10 d after opening of individual flowers (DAO) were exposed to 15°C treatment for more than 10 d (Oka et al., 1989). Therefore, plants were exposed to chilling for 2 wk beginning 8 d after anthesis of individual plants (DAA). Pots were randomized both in the phytotron and the greenhouse, and repositioned twice a week in the greenhouse and daily in the phytotron. After 2 wk of the chilling treatment, pots were returned to the greenhouse. The number of days from planting to opening of the first flower (R1, Fehr et al., 1971) and from planting to maturity (R8, when 95% of pods had mature color) were recorded for individual plants. Means of days from planting to flowering and maturity among NILs were compared with Tukey's HSD test.
Because flowering period in soybean spans across 2 wk, chilling treatment of an individual plant leads to exposure of flowers at various stages of development. Accordingly, flowers were individually labeled with the date of opening and only the flowers that opened before the chilling treatment began were used for analysis. Flowers that opened at 1 and 6 DAA were subjected to chilling stress from 7 and 2 DAO, respectively. The degree of pigmentation of each seed was scored using a pigmentation index (0 = not pigmented to 4 = severely pigmented) and a cracking index (0 = not cracked to 4 = severely cracked) as previously described (Takahashi and Abe, 1994; Takahashi, 1997). The effect of a 2-wk chilling treatment initiated at different flower stages on pigmentation and cracking was evaluated by averaging the indices of seeds obtained from flowers that were exposed to chilling stress at the same developmental stage. Pigmentation and cracking indices were subjected to analysis of variance.
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Results and discussion |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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The dominant alleles, E1 to E5, delay the transition from vegetative to reproductive growth in soybean. In this study, Harosoy-E1, Harosoy-E2, and Harosoy-E5 flowered 16, 8, and 11 d later than Harosoy (Table 2) . Harosoy-E1, Harosoy-E2, and Harosoy-E5 matured 11, 4, and 13 d later than Harosoy. Days to anthesis and maturity did not differ among Harosoy, Harosoy-e3, and Harosoy-e4. Our results are in agreement with those of McBlain and Bernard (1987) who also used Harosoy and its NILs and found the delaying effect of the dominant maturity alleles was highest in E1, intermediate in E2 and E5, and lowest in E3. Daylength at the location of this experiment may not be long enough for E3 and E4 to exhibit their delaying effect, because neither E3 nor E4 loci delayed flowering and maturity under a 12-h photoperiod (Cober et al., 1996). Chilling stress retarded maturity by 7 to 8 d in Harosoy, Harosoy-e3, Harosoy-e4, and Harosoy-E5, by 11 d in Harosoy-E1, and by 17 d in Harosoy-E2. Thus, maturity of Harosoy-E2 was strongly retarded by the chilling treatment.
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Cracked seeds were observed in plants continuously grown in the greenhouse as well as chilling-treated plants (Table 3). Kinetics of cracking from 0 to 8 DAO were different among the NILs (Fig. 2) . Early-maturing NILs, Harosoy, Harosoy-e3, and Harosoy-e4, had similar kinetics. Their cracking indices decreased with the age of flowers from 0 to 8 DAO. On the other hand, the cracking index increased slightly with the age of flowers from 0 to 5 or 7 DAO in the late-maturing NILs, Harosoy-E1, Harosoy-E2, and Harosoy-E5.
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Our study demonstrates that the NILs for five maturity genes had unique responses to low temperature in terms of seed coat pigmentation and cracking. Particularly, dominant alleles E1 and E5 possessed a marked inhibitory effect on seed coat pigmentation and cracking, while E3 did not have notable effects. The suppressing effect of E2 seemed to be weaker than those of E1 and E5. Both pigmentation and cracking were more intense in dominant alleles only at the E4 locus.
Treatments were initiated in the late-flowering NILs 10 d later than in the early-flowering ones. It is unlikely that this delay brought about any large differences in environmental conditions such as daylength and temperature. Therefore, environmental factors during pre- or post-treatments should not be a major cause of different responses among the NILs. The differences are most likely ascribable to the effects of the maturity genes themselves, although the roles of closely linked gene(s) or the remaining heterogeneity could not be completely excluded. Because the late-maturing plants were subjected to the chilling treatments at a later stage in the plants' development, the maturity genes may have indirectly affected seed coat pigmentation and cracking through differences in the plant age among NILs at the time of chilling treatment. Alternatively, maturity genes may have directly affected the seed coat deterioration. A series of physiological events leading to transition from vegetative to reproductive growth (Martínez-Zapater et al., 1994) is possibly involved in the process of seed coat pigmentation and cracking in soybean. In both cases, some of the maturity genes may help suppress the seed coat deterioration, although the underlying physiological mechanisms remain to be identified.
Takahashi and Abe (1994) performed a genetic analysis using F1 hybrids between a sensitive cultivar Kitakomachi and a tolerant cultivar Koganejiro and their F2 population after exposure to chilling stress. They found that one of the genes for tolerance was closely associated with the dominant allele for late maturity, the recessive allele of which was involved in floral induction under ILD conditions. Of six loci reported to control time of flowering and maturity in soybean, E3 and E4 are known to be involved in the response of flowering to long daylength. The recessive alleles, e3 and e4, jointly confer insensitivity to ILD (Buzzell 1971; Buzzell and Voldeng, 1980). When combined with e3 and e4, E1 markedly retards flowering under ILD relative to e1 (Cober et al., 1996). The dominant alleles, E3 and E4, had no notable influence on seed coat pigmentation. Thus, E1 may be the most likely late-maturity gene that cosegregated with the gene for tolerance in the cross between Kitakomachi and Koganejiro. However, a further study is needed to evaluate the response of E2 and E5 to ILD under the genotypes of e3e4, and the role of the other gene involved in the ILD sensitivity (Abe et al., 1998). Positive correlation between flowering date and the tolerance observed in the F2 population (Takahashi and Abe, 1994) and the NILs in this study supports that the genes for late maturity themselves were involved in the suppression of deterioration of seed coat quality.
Cracking of seed coats adversely affects external appearance and reduces their commercial value. Seed coat cracking results from separation of epidermal and hypodermal tissues, which exposes the underlying parenchyma tissue (Wolf and Baker, 1972). Based on genetic studies, Liu (1949) recognized two types of cracking, Type I with irregular cracks and Type II with net-like cracks. These cracking types occur only in a few cultivars regardless of environmental conditions. Type I cracking is controlled by genes T and I (Stewart and Wentz, 1930; Nicholas et al., 1993). Soybeans with double recessive genotypes (i and t, or i-k and t) produce severely cracked seeds, irrespective of environmental conditions, while no such symptom is observed with the other allelic combinations (Nicholas et al., 1993). Low temperature-induced cracking is also inhibited by dominant alleles T and I (Takahashi and Asanuma, 1996; Takahashi, 1997). Considering the similar inhibitory effects of these genes, low temperature-induced cracking and Type I cracking seem to be derived from one or more identical physiological mechanisms.
Most soybean cultivars occasionally exhibit seed coat cracking to some extent when exposed to adverse environmental conditions such as alternate wetting and drying at maturation (Wolf et al., 1981), chilling temperatures at flowering (Sunada and Ito, 1982; Takahashi, 1997), and pod-removal treatments (Okabe, 1996). In our experiment, cracking occurred in plants grown continuously in the greenhouse as well as in chilling-treated plants. Seed coat cracking may be an inherent tendency in soybean and it may be intensified by environmental stimuli. Under pod-removal experiments, the degree of cracking was negatively associated with time to maturity in an F2 population (Okabe, 1996) and among cultivars (Okabe et al., 1984). The relationship was also observed with the NILs for maturity genes in this study. The cracking symptoms observer under different environmental stresses may be induced through a common mechanism or mechanisms related to these maturity genes, although the underlying physiological mechanisms of seed coat cracking remain to be fully understood (Yaklich and Barla-Szabo, 1993).
Insensitivity of flowering to long daylength is an essential trait in adaptation of soybean to high latitudes with short growing seasons and long daylength. The double recessive genotype e3e4 is ILD insensitive and flowers under long daylengths (Saindon et al., 1989). Our study suggests that e3e4 may not be fully tolerant in terms of seed coat pigmentation and cracking under low temperature conditions. However, E1 and E5 may offset seed coat deterioration, although their effects under e3e4 background remain to be confirmed. `Kitamishiro' (PI 317.334A, MG1), a chilling-tolerant cultivar in terms of seed yield reduction in Hokkaido, has an allelic combination of E1e3e4 T i-i (Saindon et al., 1990). Because Kitamishiro has brown pubescence (T) and is therefore tolerant to the seed coat pigmentation, the effects of E1e3e4 on the pigmentation could not be evaluated. Kitamishiro is insensitive to long daylength and has an appropriate maturing time, indicating that E1e3e4 may be sufficiently adaptive to Hokkaido.
Genotypes with brown pubescence (T) are tolerant to the seed coat pigmentation and cracking. However, when genotypes with yellow hilum and gray pubescence (tI) are preferred for confectionery use, as in Japan, E1 and E5, singly or in combination if possible, may be useful in reducing seed coat pigmentation and cracking induced by low temperatures, when combined with the genotype of e3e4 that is adapted to high latitudes.
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ACKNOWLEDGMENTS |
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Received for publication February 2, 1999.
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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