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Postflowering photoperiod regulates vegetative growth and reproductive development of soybean
Environmental and Experimental Botany 55 (2006) 120–129
Postflowering photoperiod regulates vegetative growth and reproductive development of soybean Tianfu Hana,b,∗, Cunxiang Wua, Zhe Tongc, Rao S. Mentreddyd, Kehui Tanc, Junyi Gaib a
Key Laboratory of Crop Genetics and Breeding, Institute of Crop Sciences, The Chinese Academy of Agricultural Sciences, 12 Zhongguancun South Street, Beijing 100081, China b National Center for Soybean Improvement, Nanjing Agricultural University, Nanjing 210095, China c Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China d Department of Plant and Soil Science, Alabama A&M University, Normal, AL 35762, USA Accepted 13 October 2004
Abstract Soybean development is controlled by environmental factors, primarily photoperiod and temperature. To date, photoperiod effects on flowering have been well studied but the performances and mechanism of postflowering photoperiod responses have not been fully understood, especially for the photoperiod effects on vegetative growth after flowering. In the present study, the responses of vegetative growth and reproductive development in soybean to different postflowering photoperiod regimes were investigated in four separate experiments. Three varieties of different maturity groups (MG) including the early (Dongnong 36, MG 000), medium (Dandou 5, MG IV), and late (Zigongdongdou, MG IX) were exposed to two photoperiods, short (10, 12 h) and long (15, 16 or 18 h). The results showed that postflowering photoperiod not only regulated reproductive development but also affected vegetative growth. Even when flowers and pods were removed, short-day (SD) treatment promoted leaf senescence. The onset of leaf senescence among varieties tested appeared to be dependent on photoperiod sensitivity. Leaf senescence of the late-maturing variety of Zigongdongdou (sensitive to photoperiod) was delayed more significantly than that of the medium and early-maturing varieties (less sensitive to photoperiod). Long-day (LD) treatments delayed leaf senescence and seed maturation in the late-maturing variety of Zigongdongdou plants with only the SD-induced leaves produced before flowering. LD treatments imposed from the beginning bloom, beginning pod setting or beginning seed filling delayed leaf senescence and seed maturation of late-maturing soybean variety (Zigongdongdou). Results of night-break with red (R) and far-red (FR) light demonstrated that postflowering photoperiod responses of soybean were R/FR reversible reactions and the phytochromes seemed to be functional as receptors of photoperiod signals even after flowering. It was proposed that the regulation of photoperiod on development of soybean was effective from emergence through maturation, and the postflowering photoperiod signals were also mediated by phytochromes similar to those before flowering. The flowering reversion in late-MG soybean varieties under LD was a direct
∗
Corresponding author. Tel.: +86 10 68918784; fax: +86 10 68975212. E-mail address: [email protected] (T. Han).
0098-8472/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2004.10.006
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result of LD and was not due to secondary effect of abscission of pods and flowers. Soybean leaves not only received SD signals but also LD signals; furthermore, the LD effects reversed the SD effects and vice versa. Keywords: Soybean; Glycine max; Photoperiod; Vegetative growth; Flowering reversion; Reproductive development
© 2004 Elsevier B.V. All rights reserved.
1. Introduction The effects of photoperiod on flowering in soybean have been well documented since early 20th century (Garner and Allard, 1920, 1923; Borthwick and Parker, 1938). Photoperiod has been reported to play a major role in flowering induction (Borthwick and Parker, 1938), floral organ differentiation (Zhang et al., 2001), microsporogenesis (Nielson, 1942), postflowering development (Johnson et al., 1960; Fisher, 1963; Thomas and Raper, 1976; Raper and Thomas, 1978; Guiamet and Nakayama, 1984; Morandi et al., 1988; Han and Wang, 1995; Kantolic and Slafer, 2001), and yield formation (Mann and Jaworski, 1970; Raper and Thomas, 1978; Kantolic and Slafer, 2001) of soybean. The previous studies mainly focused on reproductive features, instead of vegetative growth. As one major factor for photosynthetic productivity of soybean, functional leaf area is determined by numbers of nodes on main stem and branches, area per leaf, and longevity of leaves (Dong, 2000). Since soybean plants continue to produce new branches and leaves after beginning bloom, and the leaves produced before flowering still work after flowering, the factors affecting the postflowering development may influence the area, functional duration of leaves and productivity of plants. Although photoperiod has been proved to be the major factor controlling the postflowering development of soybean, but little is known if photoperiod controls the growth of vegetative organs and functional duration of leaves, especially its effects at various reproductive phases. Some photoperiod-sensitive soybean varieties were shown to revert to vegetative growth following flower and pod abortion, and sprout new branches when exposure to long days (LD) after flowering, although the plants had been induced to bloom by short days (SD) before flowering (Han et al., 1998). This phenomenon was previously referred to as ‘whole plant reversion’ (Han et al., 1998), a new type of flowering reversion different from floral reversion and inflorescence rever-
sion (Battey and Lyndon, 1990; Washburn and Thomas, 2000) and was construed as evidence of persistent postflowering photoperiod responses of soybean (Han and Wang, 1998). Although the abscission of flowers and pods happened after the transfer from SD to LD conditions, it was not confirmed that LD and not flower and pod abortion was the triggering factor of vegetative growth resumption during the process of ‘whole plant reversion’. If the LD effect was the major factor causing the ‘whole plant reversion’, another question would arise: could SD-treated leaves (produced before beginning bloom) receive LD signals after the plants were transferred from SD to LD? In fact, Han et al. (1998) found that most leaves of the late-maturing soybean variety of Zigongdongdou were induced by SD before flowering and there were few new leaves produced after beginning bloom at the initial stage of postflowering LD treatment. Photoperiodic responses of soybean were shown to be persistent throughout its life cycle (Han and Wang, 1995). However, in the previous studies, there was not enough evidence to show that photoperiod was the major factor affecting the durations of podding, seed filling and maturation phases since the postflowering photoperiod treatments started at the onset of blooming (Thomas and Raper, 1976; Han and Wang, 1995), and the stages of the initial pod growth and beginning of seed filling were later in LD treatments than those in SD control, when the lowering outdoor temperature might have been the dominant factor delaying the reproductive development in LD treatment (Johnson et al., 1960; Han and Wang, 1995). The photoperiod effects on vegetative growth in these phases were even less investigated. Photoperiod-induced flowering in soybean has been found to be a red/far-red (R/FR) light reversible reaction (Parker et al., 1946; Downs, 1956), indicating that this reaction was mediated by phytochromes (for review see Thomas and Vince-Prue, 1997). Experiments also showed that the postflowering reproductive development of soybean was delayed by night-break with
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mixed light (Morandi et al., 1988; Han and Wang, 1995), indicating that the postflowering photoperiod responses of soybean shared a common mechanism with that of flowering responses. However, it is not clear if the postflowering vegetative growth and reproductive development are also R/FR reversible reactions. In the present study, four experiments were conducted with the following objectives: (i) to investigate photoperiod effects on vegetative growth after flowering; (ii) to determine whether the vegetative growth resumption in response to LD treatment was the direct effect of LD or the consequence of flower and pod abscission; (iii) to test if postflowering effects on vegetative growth and reproductive development existed in various reproductive phases of soybean; and (iv) to evaluate whether postflowering photoperiod responses were R/FR reversible and phytochrome-mediated reactions.
2. Materials and methods 2.1. Experiment 1 2.1.1. Postflowering photoperiod treatments on soybean plants with or without flower and pod removal This experiment comprised of two sub-tests. The first test was conducted at the Northeast Agricultural University campus in Harbin (45◦ 41 N, the maximum natural daylength was about 15.6 h) in 1994. Three soybean (Glycine max [L.] Merr.) varieties, Dongnong 36 (early-maturing and insensitive to photoperiod, MG 000); Dandou 5 (medium maturity and photoperiod sensitive, MG IV); and Zigongdongdou (very latematuring and highly sensitive to photoperiod, MG IX) (Han and Wang, 1996) were used as materials. Seeds were planted in pots containing soil with 5 g of (NH4)2 HPO4 on May 22 and the seedlings emerged on May 30. The plants were over-seeded and then thinned to 5 seedlings per pot after emergence according to the uniformity of plants. All plants were grown under 12 h day and 12 h night until beginning bloom (R1) (Fehr and Caviness, 1977) after which they were subjected to either 12 h (SD) or 18 h (LD) photoperiod treatments. In a given photoperiod treatment, the plants were further divided into two sub-groups with 5 pots in each. In one sub-group, flowers/pods were removed as they ap-
peared, and in another sub-group, the plants were kept intact as controls. The growth stages (Fehr and Caviness, 1977) were determined by observing individual plants at 2-day interval. The color change of the top trifoliate leaves from green to yellow was considered as an indication of leaf senescence. The plants under SD treatment were grown under natural sunshine for 12 h each day followed by 12 h in the darkness. The pots were placed on platform trucks to enable transfer of plants to dark rooms. Daylength in LD treatments was obtained by extending natural photoperiod with incandescent bulbs to 18 h. The wattage of incandescent bulbs was 60 W m−2 and the photosynthetically active radiation (PAR) above the canopy was at 50 mol m−2 s−1 when the bulbs were the only source of light. The photon flux intensity was measured with Integrating Quantum/Radiometer/Photometer, Model LI188B, LI-COR Inc., USA. The height of bulbs was adjusted as necessary to maintain uniformity of light intensity above the plant canopy. The second test was conducted at the campus of Nanjing Agricultural University in Nanjing (32◦ N, the maximum natural daylength was 14.2 h). Only the late maturing soybean variety, Zigongdongdou was used in this test. Seeds were sown on May 18 and the seedlings emerged on May 24. The experimental procedures and treatments were similar to those in the experiment conducted in Harbin (Section 2.1) except for a 15 h LD treatment instead of 18 h LD in Harbin. The facilities and procedures used for extension of photoperiod in LD treatment were the same as that described above. Both experiments used a split–split plot design with complete randomized block, where photoperiod treatments (SD and LD) were the main factor, flower and pod removal treatment as the sub-factor, and the MG as the sub–sub factor. Five replications (pots) were used in the final treatment. A combined ANOVA was performed and means were separated using Duncan’s multiple range test. 2.2. Experiment 2 2.2.1. Photoperiod treatments on soybean plants with or without removal of new branches and leaves produced after flowering An experiment was conducted at the campus of the Chinese Academy of Agricultural Sciences in
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Beijing (39◦ 54 N, the maximum daylength was about 15.0 h) in 1999 and repeated in 2000. The experiment involved photoperiod treatment with and without new branch and leaves produced after flowering using the late maturity variety Zigongdongdou. Seeds were planted on May 14, 1999 and June 25, 2000. Each pot contained 10 litre soil with 5 g of (NH4 )2 HPO4 . All plants were placed under SD (12 h) (natural sunshine) till beginning bloom (R1). After R1, half of the pots were kept under SD (12 h) and another half were transferred to LD (16 h). In a given photoperiod treatment, two sub-treatments were imposed: (1) removal of new branches/leaves produced after flowering, and (2) intact plants. Each group of plants consisted of five pots, each containing five plants. Plant growth stages were recorded at 2-day intervals as illustrated for soybean by Fehr and Caviness (1977). In sub-treatment 1, new branches/leaves were removed immediately as they appeared on the plants. The photoperiod treatments, the experimental design and procedures and data analysis were similar to those described in Section 2.1 with exceptions of the wattage of incandescent bulbs which was in this case 102 W m−2 and the photosynthetically active radiation (PAR) above the canopy was about 80 mol m−2 s−1 . 2.3. Experiment 3 2.3.1. Postflowering photoperiod treatments at various developmental phases of soybean Only Zigongdongdou was used in this experiment. In Harbin, seeds were planted in May 22, 1994, in 10-litre pots containing the same soil and fertilizer as described in Section 2.1. Plants were managed following optimum practices recommended for soybean. All plants were grown under 12 h photoperiod until beginning bloom (R1). Plants were then subjected to LD treatments of 18 h. LD treatments imposed separately from (i) beginning bloom (R1), (ii) beginning pod (R3), (iii) beginning seed (R5), (iv) full seed (R6) and (v) physiological maturity (R7). The growth stages as illustrated for soybean by Fehr and Caviness (1977) were recorded on individual plants at 2-day intervals. The photoperiod treatment, experimental design and data analysis were similar to the procedures described in Section 2.1, ex-
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cept three replications (pots) were used in each treatment. 2.4. Experiment 4 2.4.1. Light interruption of dark period with red and far-red light This experiment was conducted at the Chinese Academy of Agricultural Sciences in Beijing in 1998, using Zigongdongdou, a late-maturing variety. The seeds were planted on May 7 in 10-litre pots containing same soil and fertilizer as described in Section 2.2. Plants were grown under SD (12 h) (natural sunshine) till beginning bloom (R1 stage). After R1, all plants were transferred to LD (16 h) treatment for 12 days to induce abscission of flowers and pods. Then the plants were divided into six groups for the following respective photoperiod treatments: (i) continuous SD (10 h); (ii) SD with interruption of far-red light (FR); (iii) SD with red light then far-red light interruption (R → FR); (iv) SD with red light interruption (R); (v) SD interrupted with red light, far-red light then red light (R → FR → R), and (vi) continuous LD (16 h). Three pots with 5 plants per pot were used for each treatment. The plants were exposed to each light treatment (R or FR) for 10 min. Red light intensity above the canopy was 1.8 W m−2 and the far-red intensity was 5.5 W m−2 . The interruption treatments were imposed for 85 days. Red light with spectrum peak at 658 nm was obtained by filtering red-rich light (40 W red light lamp made by Philips, Holland) through Plexiglas 501/3 mm (Roehm & Haas, Darmstadt, Germany). Far-red light was produced by filtering 20 W farred light lamps (Toshiba, Japan) through Plexiglas PG501/3 mm and PG627/3 mm. The spectrum peak of far-red light was at 730 nm. The light intensity was measured with an YSI-Kettering Radiometer (Model YSI-65A, Yellow Springs, Ohio, USA). A YSI6551 probe was equipped with the radiometer. The R and FR light sources were installed in separate dark rooms. The pots were placed on trolleys and moved into dark chambers to impose light interruption treatments. Plant growth determination, plant care, facilities for continuous SD and LD treatments, and data analysis were similar to those described in Section 2.1 except only three replications were used.
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3. Results 3.1. Effects of postflowering photoperiod treatments and flower and pod removal on soybean development Genotypic differences on soybean development were apparent in response to photoperiod and flower/pod removal effects (Table 1). For the late maturity variety Zigongdongdou, exposure to LD delayed leaf senescence and promoted branching whether flowers and pods were artificially removed or not. Under LD, the intact plants reverted to vegetative growth similar to the plants on which flowers and pods were removed by hand. In SD treatment, the plants did not revert to vegetative growth even though flowers and pods were removed. In Harbin, the leaves of the plants in the sub-treatment with flower/pod removal within SD main treatment became yellow whereas the plants subjected to flower/pod removal under LD treatment were still in vigorous vegetative growth after the termination of the experiment in October 6 (137 days after emergence). The intact plants in SD treatment matured normally 45.3 days after the beginning bloom (R1). However, in LD treatment, the intact plants resumed vegetative growth although no flower or pod
was artificially removed. This demonstrated that vegetative growth resumption in late soybean varieties under LD was directly caused by the non-inductive photoperiod, and not due to the secondary effect of the abscission of flowers and pods. Further, continued exposure to LD may have, in fact, caused flower and pod abscission. From the above results, it appears that the postflowering photoperiod not only controls seed filling and maturation, but also affects the vegetative growth such as foliage longevity and branching. When flowers and pods removed, photoperiod can still regulate leaf senescence of plants. On the other hand, the existence of reproductive organs do promote leaf senescence and influence plant longevity, indicating that the source-sink relationship may also be involved in controlling the development rate of soybean. There was genotypic variation in photoperiod effects on leaf senescence after flowering. In this study, the early maturity variety (MG000), Dongnong 36, was also insensitive to photoperiod as evidenced by leaf senescence after the removal of flowers and pods in both SD and LD treatments. The intermediate maturity (MGIV) variety, Dandou 5, was also intermediate in its sensitivity to photoperiod in that
Table 1 Postflowering photoperiod responses of soybean plantsa with or without flowers and pods Location and year Harbin, 1994
Sub-treatment
Flowers/pods removed Control
Nanjing, 1995 a
Flowers/pods removed Control
Variety
Maturity group
Duration from R1 to R7b (d) or final performances at harvest SD (12 h)
LDc
Dongnong 36 Dandou 5 Zigongdongdou Dongnong 36 Dandou 5 Zigongdongdou
000 IV IX 000 IV IX
Near senescence (R7–R8) Near senescence (R7–R8) Leaves yellow (R7) 45.4 bd 55.8 b 49.4 b
Near senescence (R7–R8) Leaves functional Vigorous vegetative growth 48.3 a >84.0 a (not matured) >92.7 a (not matured, and the flowers and pods abscised)
Zigongdongdou
IX
Leaves yellow
Vigorous vegetative growth
Zigongdongdou
IX
Near physiological maturity (R7)
Vigorous vegetative growth
All varieties were grown under SD (12 h) up to the beginning bloom (R1) stage after which they were subjected to either SD or LD photoperiod treatment. b Growth stage as per Fehr and Caviness (1977). c LD was 18 and 15 h in Harbin and Nanjing, respectively. d The means (N = 25 plants) in a row followed by a different letter were significantly different at the 0.05 level of probability.
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Table 2 Effects of postflowering photoperiod treatments on the development of soybean variety Zigongdongdoua with or without removal of postflowering vegetative growth (Beijing, 1999b ) Treatment
Postflowering photoperiod (h)
Duration of developmental phase (d)
Final growth stage at experimental termination
R1–R3
R3–R8
Intact plants
12 16
8.6 bc 47.8 a
70.9 a Not matured
Normal maturity (R8) Flowers and pods abscised, new branches appeared and plants reverted to vegetative growth
Postflowering vegetative growth removed
12 16
7.9 b 34.3 a
75.6 a Not matured
Normal maturity (R8) Flowers and pods abscised, the leaves produced before flowering were green and thick, and senescence was slow
a
Plants were grown under SD (12 h) until flowering (R1). Data in 2000 was not shown because the R3 stage was not clearly recorded. The final growth stage at experimental termination was same as that in 1999. c The means (N = 25 plants) in a column followed by a different letter are significantly different at the 0.01 level of probability. b
leaf senescence occurred in SD treatment and not in LD treatment; Zigongdongdou, a late maturity (MGIX) variety, exhibited the strongest sensitivity to photoperiod among the three varieties. In this variety, the leaves were vigorously growing in LD treatment with or without flowers/pods removed at both locations. Interestingly, in Zigongdongdou (MGIX), a late maturity variety, the duration from R1 to R7 was shorter under SD treatment than that for MGIV (medium maturity) variety, Dandou 5 (Table 1), probably because the promoting effects of SD on the development of latematuring variety of Zigongdongdou were stronger than that of medium-maturing variety, Dandou 5. 3.2. Effects of photoperiod treatments on soybean plants with removal of new branches and leaves produced after flowering Even when new branches and leaves produced after flowering were removed and the plants had only the SD-induced leaves produced before flowering, the postflowering LD still strongly inhibited reproductive development and promoted the vegetative growth of plants. The plants continued to initiate vegetative growth even though the newly formed branches and leaves were being continuously removed. The senescence of the existed leaves was delayed by LD (Table 2). This indicates that the preflowering SDinduced leaves could receive postflowering LD signals and produce growth regulators that promote veg-
etative growth and inhibit the reproductive development after flowering. The regulators (signals) could reverse plants from the reproductive development to the vegetative growth. It is hypothesized that, at the initial stage of the reversion, the SD-induced leaves produced before flowering receive LD signals that may cause the flowering plants to revert to vegetative growth. 3.3. Effects of postflowering photoperiod treatments in various developmental phases on soybean development Table 3 shows that LD (18 h) treatments from R1 (beginning bloom), R3 (beginning pod) and R5 (beginning seed) stages significantly (P < 0.01) delayed seed filling and/or maturation of soybean variety Zigongdongdou. LD following R1 stage delayed development of R3 stage by 41.2 days compared to that of plants grown under SD treatment. LD from R3 prolonged the days from R3 to R5 (beginning seed) only slightly but had significant effects on development from R5 to R7. LD from R5 stage onwards significantly delayed maturation. The durations from R5 to R7 and from R7 to R8 in LD treatments imposed from the R3 and R5 stages were much longer than those in SD treatment. LD treatments imposed from R1 and R3 caused resumption of vegetative growth and LD beginning from R5 significantly delayed the leaf senescence. After full seed (R6), LD did not affect seed maturation, but extended leaf longevity (Table 3). The plants in LD treatment from
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Table 3 Effects of photoperiod treatments at various phases after flowering on development of soybean variety Zigongdongdou Stage when LD treatment begana
Duration of developmental phaseb R1–R3
R3–R5
R5–R7
R1–R7
R7–R8
Stageb R1
Days >47.8 ac (Only 50% plants reached R3)
–
–
–
R3
–
7.2 a
53.8 a
>90.6 a (Not matured. Most plants reversed to vegetative status) 67.0 b
R5 R6 R7 Continuous SD
– – – 6.6 b
– – – 6.5 a
40.0 b 30.9 c – 30.9 c
53.0 c 43.8 d 43.5 d 45.5 d
>24.3 a (Not matured. Upper part of plants reversed to vegetative growth) 28.2 a 4.0 bd 5.5 b 3.8 b
a
Zigongdongdou plants were placed under SD (12 h) till the stage indicated. After the stage, the plants were transferred to 18 h (LD). R1: beginning bloom; R3: beginning pod; R5: beginning seed; R6: full seed; R7: physiological maturity; R8: full maturity (Fehr and Caviness, 1977). c The means (N = 15 plants) in a column followed by a different letter are significantly different at the 0.05 level of probability. d Pods matured but plants kept some green leaves at harvest. The plants under continuous SD (control) matured normally. b
R6 had some green leaves at harvest (129 days after emergence), whereas those in SD treatment fully matured, which proved that even after seed filling, LD could play an important role in the regulation of plant development. Based on results in Table 3, it could be inferred that photoperiod effects persist throughout flowering, podding, seed filling and even maturation phases of soybean. 3.4. Effects of light interruption of dark period with combinations of red and far-red light Plants receiving the treatments of light interruption with FR and R → FR reached maturity although the interruption delayed days to maturity compared to plants in SD alone (Table 4). However, the interruption of SD with R and R → FR → R promoted vegetative growth similar to that in LD treatment. Plants reverted to vegetative growth when light regimes R, R → FR → R were imposed. The reversal effects of FR and R after flowering were similar to that before flowering (Downs, 1956), which illustrated that the postflowering photoperiod responses were also R/FR reversible and were mediated by phytochromes similar to the preflowering responses. This indicates that phytochromes are responsible for the reception of photoperiod signals throughout the whole life cycle of the soybean plants. Interruption of the dark period with different light affected the developmental rate of plants as well as
agronomic traits (Table 4). The vegetative parameters, plant height and branch number per plant in different treatments increased in the order of SD alone, FR, R → FR, R, R → FR → R, LD, but the reproductive parameters, seed yield per plant and yield components such as number of pod, seeds per plant, and 100seed weight decreased in the order of treatments SD alone, FR, R → FR, R, R → FR → R, LD. This could have been mainly due to reversal to vegetative growth and suppression of reproductive development when exposed to red light (R) as the final light exposure or LD. There was a marked effect of the R → FR treatment on branching though not as marked as that of R alone. This means that the effect of R can be reverted by subsequent exposure to FR, but reversion is not complete in the situation of the present study.
4. Discussion It was proved that postflowering photoperiod controlled many aspects of soybean development and yield formation (Johnson et al., 1960; Mann and Jaworski, 1970; Raper and Thomas, 1978; Morandi et al., 1988; Han and Wang, 1995; Kantolic and Slafer, 2001), but little attention was paid to vegetative growth of plants after flowering. In the present study, the postflowering photoperiod effects on both vegetative growth and reproductive development were investigated, and LD
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Table 4 Effects of night-break with R and FR light after flowering on maturity dates and agronomic traits of soybean variety Zigongdongdoua Treatment
Days from the beginning of night-break to R7
Plant height (cm)
Branch
Pods
SD FR R → FR R R → FR → R LD
60.6 Db 65.8 C 80.6 B >86.0 Ac >86.0 A >86.0 A
63.4 D 62.6 D 76.3 C 100.2 B 100.8 B 128.3 A
No. per plant 1.0 D 24.4 B 0.9 D 26.8 AB 4.2 C 31.0 A 6.3 B 3.1 C 6.9 B 0.1 C 9.7 A 0.0 C
Seeds
Seed weight (g per 100)
Seed yield (g per plant)
43.8 A 44.6 A 53.1 A 5.1 B 0.0 B 0.0 B
10.9 AB 11.8 A 7.0 BC 3.7 CD 0.0 D 0.0 D
4.8 A 5.2 A 5.2 A 0.7 B 0.0 B 0.0 B
a After beginning bloom (R1), all plants were transferred to LD (16 h) treatment for 12 days to induce flower abortion and pod abscission. The plants except a set of control LD (16 h) were then transferred to 10 h photoperiod. Light interruption treatments were conducted at the 7th hour of the 14 h dark period. For more information of night-break treatments see Section 2. b The means (N = 15 plants) in a column not followed by same letter were different at 0.01 level of probability. c In postflowering LD alone (16 h) and night-break treatments of R and R → FR → R, the plants did not produce any new flowers and pods. At harvest, the plants kept vegetative status and did not show any flowering features nor reached any R stage.
after flowering was found to delay the senescence of leaves, and to cause resumption of vegetative growth of soybean, especially in photoperiod-sensitive varieties, besides its inhibitive effects on reproductive development. Exposure to LD could result in both floral reversion and inflorescence reversion after SD induction in soybean (Han et al., 1998; Washburn and Thomas, 2000). Han et al. (1998) proposed a new concept of whole plant reversion. The typical performance of the whole plant reversion was that the plants reverted to vegetative growth at many nodes, and not restricted to inflorescence or floral nodes. In this study, it was found that the resumption of vegetative growth after flowering was a direct LD effect, and not a secondary effect of abscission of flowers and pods. Even the SD-induced leaves produced before flowering could receive LD signals when plants were transferred to LD after flowering, indicating that soybean leaves could receive and respond to both SD and LD signals at various developmental stages. Such responses of plants depended on a signal-mediated mechanism involving the expression of growth promoters and inhibitors. The induced state of leaves was not permanent, but reversible when exposed to LD, even after floral commitment. Previous studies by Han and Wang (1995) proved that the responses of soybean to daylength after flowering are typical of photoperiodically controlled phenomena. Their results also showed that postflow-
ering photoperiod responses exist in various varieties belonging to different maturity groups (Han and Wang, 1996). The differences in the length of reproductive period among varieties with similar flowering dates and different maturity dates are positively correlated with the variation in postflowering photoperiod sensitivity (Han and Gai, 1998). Since the postflowering photoperiod treatments in the previous studies were imposed from beginning bloom, it was not fully proved if photoperiod was the major environmental factor controlling the development of soybean plants at middle and late reproductive phases, i.e., seed filling and maturation. Results from the present study showed that LD treatments at flowering, podding and seed filling stages all promoted the vegetative growth and inhibited reproductive development. In LD treatment, leaf senescence was delayed even when the LD treatment was imposed from full seed (R6), confirming the previous hypothesis (Han and Wang, 1995), which postulated that the photoperiod effects exist throughout the life cycle of the soybean plants. The presence of a phytochrome-mediated phenomenon is evidenced in the photoreaction that can be induced by exposure to red light and reversed by following far-red light exposure during the dark period (for review see Thomas and Vince-Prue, 1997). Parker et al. (1946) and Downs (1956) found that nightbreak with red light inhibits flowering of soybean. In the present study, it was found that effects of night-break after flowering also depend on whether R or FR is given
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as the final exposure in a series of R and FR light interruptions of the dark period. This phenomenon is similar to that reported for other Pfr-mediated responses (for review see Thomas and Vince-Prue, 1997). Final exposure to red light delayed or inhibited the reproductive development of Zigongdongdou, and the effects of red light could be reversed by exposure to far-red light. It is confirmed that the response of soybean to postflowering daylength is a typical phenomenon of photoperiodism. Based on previous (Han and Wang, 1995) and current results, it can be concluded that photoperiod responses of soybean exist from emergence to maturation, and phytochromes are the receptors of photoperiod signals throughout the whole life cycle of soybean. That is, soybean has the similar signal receiving mechanism before and after flowering. Leaves were proved to be the receivers of photoperiod signals that regulated reproductive development of soybean and other plants (for review see Thomas and Vince-Prue, 1997). However, it has not been well understood that if the reproductive organs play any roles in leaf growth and senescence, and other aspects of vegetative growth. Leopold et al. (1959) found that removal of the developing flowers and fruits delayed leaf senescence of plants. Caffaro and Nakayama (1988) proved that the removal of flowers affected the vegetative activity of the main stem terminal buds. Leopold et al. (1959) suggested that reproductive development might result in the diversion of mobilized reserve food materials towards the developing flowers and fruits. Sinclair and deWit (1975, 1976) proposed that the competitiveness of the seeds as sink abbreviates the period of seed filling by the onset of leaf senescence due to nitrogen stress. The results of the present study confirmed that pods (seeds) do promote the senescence of soybean leaves but also that photoperiod could regulate leaf senescence directly. It was proposed that, under inductive photoperiod conditions, leaves produce developmentpromoting substances including ABA (Brenner, 1987; Han and Gai, 1999), and result in the relocation of growth centers to reproductive organs in plants. The pods (seeds) could promote leaf senescence and maturation of whole plants, which may be exacerbated by nitrogen stress (Sinclair and deWit, 1975, 1976) and possibly by recycling of ABA back to leaves (Han and Gai, 1999). At the initial stage of the above pathway, SD signal received by leaves could be the triggering factor.
5. Conclusion Photoperiod is the major environmental factor regulating the postflowering development of soybean. The results in this paper showed that postflowering photoperiod not only controlled reproductive development of soybean, but also regulated leaf senescence, branching and other aspects of vegetative growth. Exposure to LD at flowering stage induced resumption of vegetative growth and abscission of flowers and pods in photoperiod-sensitive varieties. This phenomenon was previously referred to as ‘whole plant reversion’. Further studies in this paper indicated that the resumption of vegetative growth was the direct result of LD, instead of the secondary effects of abortion and abscission of reproductive organs. During the reversion process, the SD-treated leaves produced before flowering were the receiving organs of postflowering LD signals, showing that SD-treated leaves were functional under LD, and their physiological status was reversible. Results also proved that photoperiod effects on both vegetative growth and reproductive development persisted at all reproductive stages of soybean development, from the onset of flowering to seed filling and maturation. Night-break experiment demonstrated that postflowering photoperiod responses were R/FR reversible reactions and seemed to be mediated by phytochromes. The evidences obtained in the present studies further proved that the photoperiod responses were persistent throughout the whole life cycle of soybean.
Acknowledgements We thank Drs. Yonghua Yang, Yangnian Bai and Shikui Song for their comments on this manuscript; Dongxing Zhou, Chongbin Zhang, Shiwen Wang, Yuxi Shan, Haiqing Xu for plant care. We are grateful for detailed revision and helpful comments from two anonymous reviewers. This work was supported by the grants from the National Natural Science Foundation of China (30070456, 30070076), State Key Basic Research and Development Plan of China (2002CB111301), Beijing Natural Science Foundation (6012018), and the National High Technology Research and Development Program of China (2003AA207060-2).
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References Battey, N.H., Lyndon, R.F., 1990. Reversion of flowering. Bot. Rev. 56, 162–189. Borthwick, H.A., Parker, M.W., 1938. Influence of photoperiods upon the differentiation of meristems and the blossoming of Biloxi soybeans. Bot. Gaz. (Chicago) 99, 825–839. Brenner, M.L., 1987. The role of hormones in photosynthate partitioning and seed filling. In: Davies, P.J. (Ed.), Plant Hormones and their Role in Plant Growth and Development. Martinus Nijhoff Publishers, Dordrecht, pp. 474–493. Caffaro, S.V., Nakayama, F., 1988. Vegetative activity of the main stem terminal bud under photoperiod and flower removal treatments in soybean. Aust. J. Plant Physiol. 115, 159–166. Dong, Z., 2000. Soybean Yield Physiology. China Agricultural Press, Beijing, pp. 29–62, (In Chinese). Downs, R.J., 1956. Photoreversibility of flower initiation. Plant Physiol. 31, 279–284. Fehr, W.R., Caviness, C.E., 1977. Stages of soybean development. Spec. Rep. No. 80, Iowa State University, Ames, IA, USA. Fisher, J.E., 1963. The effect of short days on fruitset as distinct from flower formation in soybeans. Can. J. Bot. 41, 871– 873. Garner, W.W., Allard, H.A., 1920. Effect of the relative length of day and night and other factors of the environment on growth and reproduction in plants. J. Agric. Res. (Washington, DC) 18, 553–606. Garner, W.W., Allard, H.A., 1923. Further studies in photoperiodism, the response of the plant to relative length of day and night. J. Agric. Res. (Washington, DC) 23, 871–920. Guiamet, J.J., Nakayama, F., 1984. The effects of long days upon reproductive growth in soybeans, Glycine max, L. Merr. cv. Williams. Japan J. Crop Sci. 53, 35–40. Han, T., Gai, J., 1998. Postflowering photoperiod response of soybean varieties with similar flowering dates and different maturity dates. Soybean Genet. Newslett. 25, 48–49. Han, T., Gai, J., 1999. Phytohormonal analysis of some photoperiod effects in soybean. Acta Agron. Sinica 25, 349–355 (In Chinese, with English abstract). Han, T., Gai, J., Wang, J., Zhou, D., 1998. Discovery of flowering reversions in soybean plant. Acta Agron. Sinica 24, 168–171 (In Chinese, with English abstract).
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Han, T., Wang, J., 1995. Studies on the postflowering photoperiodic responses in soybean. Acta Botan. Sinica 37, 863–869 (In Chinese, with English abstract). Han, T., Wang, J., 1996. A study on the response of different ecotypes of Chinese soybeans to postflowering photoperiod. Acta Agron. Sinica 22, 20–26 (In Chinese, with English abstract). Johnson, H.W., Borthwick, H.A., Leffel, R.C., 1960. Effect of photoperiod and time of planting on rates of development of the soybean in various stages of the life cycle. Bot. Gaz. (Chicago) 122, 77–95. Kantolic, A.G., Slafer, G.A., 2001. Photoperiod sensitivity after flowering and seed number determination in indeterminate soybean cultivars. Field Crops Res. 72, 109–118. Leopold, A.C., Niedergang-Kamien, E., Janick, J., 1959. Experimental modification of plant senescence. Plant Physiol. 34, 570–573. Mann, J.D., Jaworski, E.G., 1970. Comparison of stresses which may limit soybean yield. Crop Sci. 10, 620–624. Morandi, E.N., Casano, L.M., Reggiardo, L.M., 1988. Postflowering photoperiodic effect on reproductive efficiency and seed growth in soybean. Field Crops Res. 18, 227–241. Nielson, C.S., 1942. Effects of photoperiod on microsporogenesis in Biloxi soybean. Bot. Gaz. (Chicago) 104, 99–106. Parker, M.W., Hendricks, S.B., Borthwick, H.A., Scully, N.J., 1946. Action spectrum for the photoperiodic control of floral initiation of short-day plants. Bot. Gaz. (Chicago) 108, 1–26. Raper Jr., C.D., Thomas, J.F., 1978. Photoperiodic alteration of dry matter partitioning and seed yield in soybean. Crop Sci. 18, 654–656. Sinclair, T.R., deWit, C.T., 1975. Comparative analysis of photosynthate and nitrogen requirement in the production of seed by various crops. Science 189, 565–567. Sinclair, T.R., deWit, C.T., 1976. Analysis of the carbon and nitrogen limitations to soybean yield. Agron. J. 68, 319–324. Thomas, F.J., Raper Jr., C.D., 1976. Photoperiodic control of seed filling for soybeans. Crop Sci. 16, 667–672. Thomas, B., Vince-Prue, D., 1997. Photoperiodism in Plants. Academic Press, San Diego, CA, USA, pp. 63–84. Washburn, C.F., Thomas, J.F., 2000. Reversion of flowering in Glycine max (Fabaceae). Amer. J. Bot. 87, 1425–1438. Zhang, L., Wang, R., Hesketh, J.D., 2001. Effects of photoperiod on growth and development of soybean floral bud in different maturity. Agron. J. 93, 944–948.
Postflowering photoperiod regulates vegetative growth and reproductive development of soybean Tianfu Hana,b,∗, Cunxiang Wua, Zhe Tongc, Rao S. Mentreddyd, Kehui Tanc, Junyi Gaib a
Key Laboratory of Crop Genetics and Breeding, Institute of Crop Sciences, The Chinese Academy of Agricultural Sciences, 12 Zhongguancun South Street, Beijing 100081, China b National Center for Soybean Improvement, Nanjing Agricultural University, Nanjing 210095, China c Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China d Department of Plant and Soil Science, Alabama A&M University, Normal, AL 35762, USA Accepted 13 October 2004
Abstract Soybean development is controlled by environmental factors, primarily photoperiod and temperature. To date, photoperiod effects on flowering have been well studied but the performances and mechanism of postflowering photoperiod responses have not been fully understood, especially for the photoperiod effects on vegetative growth after flowering. In the present study, the responses of vegetative growth and reproductive development in soybean to different postflowering photoperiod regimes were investigated in four separate experiments. Three varieties of different maturity groups (MG) including the early (Dongnong 36, MG 000), medium (Dandou 5, MG IV), and late (Zigongdongdou, MG IX) were exposed to two photoperiods, short (10, 12 h) and long (15, 16 or 18 h). The results showed that postflowering photoperiod not only regulated reproductive development but also affected vegetative growth. Even when flowers and pods were removed, short-day (SD) treatment promoted leaf senescence. The onset of leaf senescence among varieties tested appeared to be dependent on photoperiod sensitivity. Leaf senescence of the late-maturing variety of Zigongdongdou (sensitive to photoperiod) was delayed more significantly than that of the medium and early-maturing varieties (less sensitive to photoperiod). Long-day (LD) treatments delayed leaf senescence and seed maturation in the late-maturing variety of Zigongdongdou plants with only the SD-induced leaves produced before flowering. LD treatments imposed from the beginning bloom, beginning pod setting or beginning seed filling delayed leaf senescence and seed maturation of late-maturing soybean variety (Zigongdongdou). Results of night-break with red (R) and far-red (FR) light demonstrated that postflowering photoperiod responses of soybean were R/FR reversible reactions and the phytochromes seemed to be functional as receptors of photoperiod signals even after flowering. It was proposed that the regulation of photoperiod on development of soybean was effective from emergence through maturation, and the postflowering photoperiod signals were also mediated by phytochromes similar to those before flowering. The flowering reversion in late-MG soybean varieties under LD was a direct
∗
Corresponding author. Tel.: +86 10 68918784; fax: +86 10 68975212. E-mail address: [email protected] (T. Han).
0098-8472/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2004.10.006
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result of LD and was not due to secondary effect of abscission of pods and flowers. Soybean leaves not only received SD signals but also LD signals; furthermore, the LD effects reversed the SD effects and vice versa. Keywords: Soybean; Glycine max; Photoperiod; Vegetative growth; Flowering reversion; Reproductive development
© 2004 Elsevier B.V. All rights reserved.
1. Introduction The effects of photoperiod on flowering in soybean have been well documented since early 20th century (Garner and Allard, 1920, 1923; Borthwick and Parker, 1938). Photoperiod has been reported to play a major role in flowering induction (Borthwick and Parker, 1938), floral organ differentiation (Zhang et al., 2001), microsporogenesis (Nielson, 1942), postflowering development (Johnson et al., 1960; Fisher, 1963; Thomas and Raper, 1976; Raper and Thomas, 1978; Guiamet and Nakayama, 1984; Morandi et al., 1988; Han and Wang, 1995; Kantolic and Slafer, 2001), and yield formation (Mann and Jaworski, 1970; Raper and Thomas, 1978; Kantolic and Slafer, 2001) of soybean. The previous studies mainly focused on reproductive features, instead of vegetative growth. As one major factor for photosynthetic productivity of soybean, functional leaf area is determined by numbers of nodes on main stem and branches, area per leaf, and longevity of leaves (Dong, 2000). Since soybean plants continue to produce new branches and leaves after beginning bloom, and the leaves produced before flowering still work after flowering, the factors affecting the postflowering development may influence the area, functional duration of leaves and productivity of plants. Although photoperiod has been proved to be the major factor controlling the postflowering development of soybean, but little is known if photoperiod controls the growth of vegetative organs and functional duration of leaves, especially its effects at various reproductive phases. Some photoperiod-sensitive soybean varieties were shown to revert to vegetative growth following flower and pod abortion, and sprout new branches when exposure to long days (LD) after flowering, although the plants had been induced to bloom by short days (SD) before flowering (Han et al., 1998). This phenomenon was previously referred to as ‘whole plant reversion’ (Han et al., 1998), a new type of flowering reversion different from floral reversion and inflorescence rever-
sion (Battey and Lyndon, 1990; Washburn and Thomas, 2000) and was construed as evidence of persistent postflowering photoperiod responses of soybean (Han and Wang, 1998). Although the abscission of flowers and pods happened after the transfer from SD to LD conditions, it was not confirmed that LD and not flower and pod abortion was the triggering factor of vegetative growth resumption during the process of ‘whole plant reversion’. If the LD effect was the major factor causing the ‘whole plant reversion’, another question would arise: could SD-treated leaves (produced before beginning bloom) receive LD signals after the plants were transferred from SD to LD? In fact, Han et al. (1998) found that most leaves of the late-maturing soybean variety of Zigongdongdou were induced by SD before flowering and there were few new leaves produced after beginning bloom at the initial stage of postflowering LD treatment. Photoperiodic responses of soybean were shown to be persistent throughout its life cycle (Han and Wang, 1995). However, in the previous studies, there was not enough evidence to show that photoperiod was the major factor affecting the durations of podding, seed filling and maturation phases since the postflowering photoperiod treatments started at the onset of blooming (Thomas and Raper, 1976; Han and Wang, 1995), and the stages of the initial pod growth and beginning of seed filling were later in LD treatments than those in SD control, when the lowering outdoor temperature might have been the dominant factor delaying the reproductive development in LD treatment (Johnson et al., 1960; Han and Wang, 1995). The photoperiod effects on vegetative growth in these phases were even less investigated. Photoperiod-induced flowering in soybean has been found to be a red/far-red (R/FR) light reversible reaction (Parker et al., 1946; Downs, 1956), indicating that this reaction was mediated by phytochromes (for review see Thomas and Vince-Prue, 1997). Experiments also showed that the postflowering reproductive development of soybean was delayed by night-break with
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mixed light (Morandi et al., 1988; Han and Wang, 1995), indicating that the postflowering photoperiod responses of soybean shared a common mechanism with that of flowering responses. However, it is not clear if the postflowering vegetative growth and reproductive development are also R/FR reversible reactions. In the present study, four experiments were conducted with the following objectives: (i) to investigate photoperiod effects on vegetative growth after flowering; (ii) to determine whether the vegetative growth resumption in response to LD treatment was the direct effect of LD or the consequence of flower and pod abscission; (iii) to test if postflowering effects on vegetative growth and reproductive development existed in various reproductive phases of soybean; and (iv) to evaluate whether postflowering photoperiod responses were R/FR reversible and phytochrome-mediated reactions.
2. Materials and methods 2.1. Experiment 1 2.1.1. Postflowering photoperiod treatments on soybean plants with or without flower and pod removal This experiment comprised of two sub-tests. The first test was conducted at the Northeast Agricultural University campus in Harbin (45◦ 41 N, the maximum natural daylength was about 15.6 h) in 1994. Three soybean (Glycine max [L.] Merr.) varieties, Dongnong 36 (early-maturing and insensitive to photoperiod, MG 000); Dandou 5 (medium maturity and photoperiod sensitive, MG IV); and Zigongdongdou (very latematuring and highly sensitive to photoperiod, MG IX) (Han and Wang, 1996) were used as materials. Seeds were planted in pots containing soil with 5 g of (NH4)2 HPO4 on May 22 and the seedlings emerged on May 30. The plants were over-seeded and then thinned to 5 seedlings per pot after emergence according to the uniformity of plants. All plants were grown under 12 h day and 12 h night until beginning bloom (R1) (Fehr and Caviness, 1977) after which they were subjected to either 12 h (SD) or 18 h (LD) photoperiod treatments. In a given photoperiod treatment, the plants were further divided into two sub-groups with 5 pots in each. In one sub-group, flowers/pods were removed as they ap-
peared, and in another sub-group, the plants were kept intact as controls. The growth stages (Fehr and Caviness, 1977) were determined by observing individual plants at 2-day interval. The color change of the top trifoliate leaves from green to yellow was considered as an indication of leaf senescence. The plants under SD treatment were grown under natural sunshine for 12 h each day followed by 12 h in the darkness. The pots were placed on platform trucks to enable transfer of plants to dark rooms. Daylength in LD treatments was obtained by extending natural photoperiod with incandescent bulbs to 18 h. The wattage of incandescent bulbs was 60 W m−2 and the photosynthetically active radiation (PAR) above the canopy was at 50 mol m−2 s−1 when the bulbs were the only source of light. The photon flux intensity was measured with Integrating Quantum/Radiometer/Photometer, Model LI188B, LI-COR Inc., USA. The height of bulbs was adjusted as necessary to maintain uniformity of light intensity above the plant canopy. The second test was conducted at the campus of Nanjing Agricultural University in Nanjing (32◦ N, the maximum natural daylength was 14.2 h). Only the late maturing soybean variety, Zigongdongdou was used in this test. Seeds were sown on May 18 and the seedlings emerged on May 24. The experimental procedures and treatments were similar to those in the experiment conducted in Harbin (Section 2.1) except for a 15 h LD treatment instead of 18 h LD in Harbin. The facilities and procedures used for extension of photoperiod in LD treatment were the same as that described above. Both experiments used a split–split plot design with complete randomized block, where photoperiod treatments (SD and LD) were the main factor, flower and pod removal treatment as the sub-factor, and the MG as the sub–sub factor. Five replications (pots) were used in the final treatment. A combined ANOVA was performed and means were separated using Duncan’s multiple range test. 2.2. Experiment 2 2.2.1. Photoperiod treatments on soybean plants with or without removal of new branches and leaves produced after flowering An experiment was conducted at the campus of the Chinese Academy of Agricultural Sciences in
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Beijing (39◦ 54 N, the maximum daylength was about 15.0 h) in 1999 and repeated in 2000. The experiment involved photoperiod treatment with and without new branch and leaves produced after flowering using the late maturity variety Zigongdongdou. Seeds were planted on May 14, 1999 and June 25, 2000. Each pot contained 10 litre soil with 5 g of (NH4 )2 HPO4 . All plants were placed under SD (12 h) (natural sunshine) till beginning bloom (R1). After R1, half of the pots were kept under SD (12 h) and another half were transferred to LD (16 h). In a given photoperiod treatment, two sub-treatments were imposed: (1) removal of new branches/leaves produced after flowering, and (2) intact plants. Each group of plants consisted of five pots, each containing five plants. Plant growth stages were recorded at 2-day intervals as illustrated for soybean by Fehr and Caviness (1977). In sub-treatment 1, new branches/leaves were removed immediately as they appeared on the plants. The photoperiod treatments, the experimental design and procedures and data analysis were similar to those described in Section 2.1 with exceptions of the wattage of incandescent bulbs which was in this case 102 W m−2 and the photosynthetically active radiation (PAR) above the canopy was about 80 mol m−2 s−1 . 2.3. Experiment 3 2.3.1. Postflowering photoperiod treatments at various developmental phases of soybean Only Zigongdongdou was used in this experiment. In Harbin, seeds were planted in May 22, 1994, in 10-litre pots containing the same soil and fertilizer as described in Section 2.1. Plants were managed following optimum practices recommended for soybean. All plants were grown under 12 h photoperiod until beginning bloom (R1). Plants were then subjected to LD treatments of 18 h. LD treatments imposed separately from (i) beginning bloom (R1), (ii) beginning pod (R3), (iii) beginning seed (R5), (iv) full seed (R6) and (v) physiological maturity (R7). The growth stages as illustrated for soybean by Fehr and Caviness (1977) were recorded on individual plants at 2-day intervals. The photoperiod treatment, experimental design and data analysis were similar to the procedures described in Section 2.1, ex-
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cept three replications (pots) were used in each treatment. 2.4. Experiment 4 2.4.1. Light interruption of dark period with red and far-red light This experiment was conducted at the Chinese Academy of Agricultural Sciences in Beijing in 1998, using Zigongdongdou, a late-maturing variety. The seeds were planted on May 7 in 10-litre pots containing same soil and fertilizer as described in Section 2.2. Plants were grown under SD (12 h) (natural sunshine) till beginning bloom (R1 stage). After R1, all plants were transferred to LD (16 h) treatment for 12 days to induce abscission of flowers and pods. Then the plants were divided into six groups for the following respective photoperiod treatments: (i) continuous SD (10 h); (ii) SD with interruption of far-red light (FR); (iii) SD with red light then far-red light interruption (R → FR); (iv) SD with red light interruption (R); (v) SD interrupted with red light, far-red light then red light (R → FR → R), and (vi) continuous LD (16 h). Three pots with 5 plants per pot were used for each treatment. The plants were exposed to each light treatment (R or FR) for 10 min. Red light intensity above the canopy was 1.8 W m−2 and the far-red intensity was 5.5 W m−2 . The interruption treatments were imposed for 85 days. Red light with spectrum peak at 658 nm was obtained by filtering red-rich light (40 W red light lamp made by Philips, Holland) through Plexiglas 501/3 mm (Roehm & Haas, Darmstadt, Germany). Far-red light was produced by filtering 20 W farred light lamps (Toshiba, Japan) through Plexiglas PG501/3 mm and PG627/3 mm. The spectrum peak of far-red light was at 730 nm. The light intensity was measured with an YSI-Kettering Radiometer (Model YSI-65A, Yellow Springs, Ohio, USA). A YSI6551 probe was equipped with the radiometer. The R and FR light sources were installed in separate dark rooms. The pots were placed on trolleys and moved into dark chambers to impose light interruption treatments. Plant growth determination, plant care, facilities for continuous SD and LD treatments, and data analysis were similar to those described in Section 2.1 except only three replications were used.
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3. Results 3.1. Effects of postflowering photoperiod treatments and flower and pod removal on soybean development Genotypic differences on soybean development were apparent in response to photoperiod and flower/pod removal effects (Table 1). For the late maturity variety Zigongdongdou, exposure to LD delayed leaf senescence and promoted branching whether flowers and pods were artificially removed or not. Under LD, the intact plants reverted to vegetative growth similar to the plants on which flowers and pods were removed by hand. In SD treatment, the plants did not revert to vegetative growth even though flowers and pods were removed. In Harbin, the leaves of the plants in the sub-treatment with flower/pod removal within SD main treatment became yellow whereas the plants subjected to flower/pod removal under LD treatment were still in vigorous vegetative growth after the termination of the experiment in October 6 (137 days after emergence). The intact plants in SD treatment matured normally 45.3 days after the beginning bloom (R1). However, in LD treatment, the intact plants resumed vegetative growth although no flower or pod
was artificially removed. This demonstrated that vegetative growth resumption in late soybean varieties under LD was directly caused by the non-inductive photoperiod, and not due to the secondary effect of the abscission of flowers and pods. Further, continued exposure to LD may have, in fact, caused flower and pod abscission. From the above results, it appears that the postflowering photoperiod not only controls seed filling and maturation, but also affects the vegetative growth such as foliage longevity and branching. When flowers and pods removed, photoperiod can still regulate leaf senescence of plants. On the other hand, the existence of reproductive organs do promote leaf senescence and influence plant longevity, indicating that the source-sink relationship may also be involved in controlling the development rate of soybean. There was genotypic variation in photoperiod effects on leaf senescence after flowering. In this study, the early maturity variety (MG000), Dongnong 36, was also insensitive to photoperiod as evidenced by leaf senescence after the removal of flowers and pods in both SD and LD treatments. The intermediate maturity (MGIV) variety, Dandou 5, was also intermediate in its sensitivity to photoperiod in that
Table 1 Postflowering photoperiod responses of soybean plantsa with or without flowers and pods Location and year Harbin, 1994
Sub-treatment
Flowers/pods removed Control
Nanjing, 1995 a
Flowers/pods removed Control
Variety
Maturity group
Duration from R1 to R7b (d) or final performances at harvest SD (12 h)
LDc
Dongnong 36 Dandou 5 Zigongdongdou Dongnong 36 Dandou 5 Zigongdongdou
000 IV IX 000 IV IX
Near senescence (R7–R8) Near senescence (R7–R8) Leaves yellow (R7) 45.4 bd 55.8 b 49.4 b
Near senescence (R7–R8) Leaves functional Vigorous vegetative growth 48.3 a >84.0 a (not matured) >92.7 a (not matured, and the flowers and pods abscised)
Zigongdongdou
IX
Leaves yellow
Vigorous vegetative growth
Zigongdongdou
IX
Near physiological maturity (R7)
Vigorous vegetative growth
All varieties were grown under SD (12 h) up to the beginning bloom (R1) stage after which they were subjected to either SD or LD photoperiod treatment. b Growth stage as per Fehr and Caviness (1977). c LD was 18 and 15 h in Harbin and Nanjing, respectively. d The means (N = 25 plants) in a row followed by a different letter were significantly different at the 0.05 level of probability.
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Table 2 Effects of postflowering photoperiod treatments on the development of soybean variety Zigongdongdoua with or without removal of postflowering vegetative growth (Beijing, 1999b ) Treatment
Postflowering photoperiod (h)
Duration of developmental phase (d)
Final growth stage at experimental termination
R1–R3
R3–R8
Intact plants
12 16
8.6 bc 47.8 a
70.9 a Not matured
Normal maturity (R8) Flowers and pods abscised, new branches appeared and plants reverted to vegetative growth
Postflowering vegetative growth removed
12 16
7.9 b 34.3 a
75.6 a Not matured
Normal maturity (R8) Flowers and pods abscised, the leaves produced before flowering were green and thick, and senescence was slow
a
Plants were grown under SD (12 h) until flowering (R1). Data in 2000 was not shown because the R3 stage was not clearly recorded. The final growth stage at experimental termination was same as that in 1999. c The means (N = 25 plants) in a column followed by a different letter are significantly different at the 0.01 level of probability. b
leaf senescence occurred in SD treatment and not in LD treatment; Zigongdongdou, a late maturity (MGIX) variety, exhibited the strongest sensitivity to photoperiod among the three varieties. In this variety, the leaves were vigorously growing in LD treatment with or without flowers/pods removed at both locations. Interestingly, in Zigongdongdou (MGIX), a late maturity variety, the duration from R1 to R7 was shorter under SD treatment than that for MGIV (medium maturity) variety, Dandou 5 (Table 1), probably because the promoting effects of SD on the development of latematuring variety of Zigongdongdou were stronger than that of medium-maturing variety, Dandou 5. 3.2. Effects of photoperiod treatments on soybean plants with removal of new branches and leaves produced after flowering Even when new branches and leaves produced after flowering were removed and the plants had only the SD-induced leaves produced before flowering, the postflowering LD still strongly inhibited reproductive development and promoted the vegetative growth of plants. The plants continued to initiate vegetative growth even though the newly formed branches and leaves were being continuously removed. The senescence of the existed leaves was delayed by LD (Table 2). This indicates that the preflowering SDinduced leaves could receive postflowering LD signals and produce growth regulators that promote veg-
etative growth and inhibit the reproductive development after flowering. The regulators (signals) could reverse plants from the reproductive development to the vegetative growth. It is hypothesized that, at the initial stage of the reversion, the SD-induced leaves produced before flowering receive LD signals that may cause the flowering plants to revert to vegetative growth. 3.3. Effects of postflowering photoperiod treatments in various developmental phases on soybean development Table 3 shows that LD (18 h) treatments from R1 (beginning bloom), R3 (beginning pod) and R5 (beginning seed) stages significantly (P < 0.01) delayed seed filling and/or maturation of soybean variety Zigongdongdou. LD following R1 stage delayed development of R3 stage by 41.2 days compared to that of plants grown under SD treatment. LD from R3 prolonged the days from R3 to R5 (beginning seed) only slightly but had significant effects on development from R5 to R7. LD from R5 stage onwards significantly delayed maturation. The durations from R5 to R7 and from R7 to R8 in LD treatments imposed from the R3 and R5 stages were much longer than those in SD treatment. LD treatments imposed from R1 and R3 caused resumption of vegetative growth and LD beginning from R5 significantly delayed the leaf senescence. After full seed (R6), LD did not affect seed maturation, but extended leaf longevity (Table 3). The plants in LD treatment from
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Table 3 Effects of photoperiod treatments at various phases after flowering on development of soybean variety Zigongdongdou Stage when LD treatment begana
Duration of developmental phaseb R1–R3
R3–R5
R5–R7
R1–R7
R7–R8
Stageb R1
Days >47.8 ac (Only 50% plants reached R3)
–
–
–
R3
–
7.2 a
53.8 a
>90.6 a (Not matured. Most plants reversed to vegetative status) 67.0 b
R5 R6 R7 Continuous SD
– – – 6.6 b
– – – 6.5 a
40.0 b 30.9 c – 30.9 c
53.0 c 43.8 d 43.5 d 45.5 d
>24.3 a (Not matured. Upper part of plants reversed to vegetative growth) 28.2 a 4.0 bd 5.5 b 3.8 b
a
Zigongdongdou plants were placed under SD (12 h) till the stage indicated. After the stage, the plants were transferred to 18 h (LD). R1: beginning bloom; R3: beginning pod; R5: beginning seed; R6: full seed; R7: physiological maturity; R8: full maturity (Fehr and Caviness, 1977). c The means (N = 15 plants) in a column followed by a different letter are significantly different at the 0.05 level of probability. d Pods matured but plants kept some green leaves at harvest. The plants under continuous SD (control) matured normally. b
R6 had some green leaves at harvest (129 days after emergence), whereas those in SD treatment fully matured, which proved that even after seed filling, LD could play an important role in the regulation of plant development. Based on results in Table 3, it could be inferred that photoperiod effects persist throughout flowering, podding, seed filling and even maturation phases of soybean. 3.4. Effects of light interruption of dark period with combinations of red and far-red light Plants receiving the treatments of light interruption with FR and R → FR reached maturity although the interruption delayed days to maturity compared to plants in SD alone (Table 4). However, the interruption of SD with R and R → FR → R promoted vegetative growth similar to that in LD treatment. Plants reverted to vegetative growth when light regimes R, R → FR → R were imposed. The reversal effects of FR and R after flowering were similar to that before flowering (Downs, 1956), which illustrated that the postflowering photoperiod responses were also R/FR reversible and were mediated by phytochromes similar to the preflowering responses. This indicates that phytochromes are responsible for the reception of photoperiod signals throughout the whole life cycle of the soybean plants. Interruption of the dark period with different light affected the developmental rate of plants as well as
agronomic traits (Table 4). The vegetative parameters, plant height and branch number per plant in different treatments increased in the order of SD alone, FR, R → FR, R, R → FR → R, LD, but the reproductive parameters, seed yield per plant and yield components such as number of pod, seeds per plant, and 100seed weight decreased in the order of treatments SD alone, FR, R → FR, R, R → FR → R, LD. This could have been mainly due to reversal to vegetative growth and suppression of reproductive development when exposed to red light (R) as the final light exposure or LD. There was a marked effect of the R → FR treatment on branching though not as marked as that of R alone. This means that the effect of R can be reverted by subsequent exposure to FR, but reversion is not complete in the situation of the present study.
4. Discussion It was proved that postflowering photoperiod controlled many aspects of soybean development and yield formation (Johnson et al., 1960; Mann and Jaworski, 1970; Raper and Thomas, 1978; Morandi et al., 1988; Han and Wang, 1995; Kantolic and Slafer, 2001), but little attention was paid to vegetative growth of plants after flowering. In the present study, the postflowering photoperiod effects on both vegetative growth and reproductive development were investigated, and LD
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Table 4 Effects of night-break with R and FR light after flowering on maturity dates and agronomic traits of soybean variety Zigongdongdoua Treatment
Days from the beginning of night-break to R7
Plant height (cm)
Branch
Pods
SD FR R → FR R R → FR → R LD
60.6 Db 65.8 C 80.6 B >86.0 Ac >86.0 A >86.0 A
63.4 D 62.6 D 76.3 C 100.2 B 100.8 B 128.3 A
No. per plant 1.0 D 24.4 B 0.9 D 26.8 AB 4.2 C 31.0 A 6.3 B 3.1 C 6.9 B 0.1 C 9.7 A 0.0 C
Seeds
Seed weight (g per 100)
Seed yield (g per plant)
43.8 A 44.6 A 53.1 A 5.1 B 0.0 B 0.0 B
10.9 AB 11.8 A 7.0 BC 3.7 CD 0.0 D 0.0 D
4.8 A 5.2 A 5.2 A 0.7 B 0.0 B 0.0 B
a After beginning bloom (R1), all plants were transferred to LD (16 h) treatment for 12 days to induce flower abortion and pod abscission. The plants except a set of control LD (16 h) were then transferred to 10 h photoperiod. Light interruption treatments were conducted at the 7th hour of the 14 h dark period. For more information of night-break treatments see Section 2. b The means (N = 15 plants) in a column not followed by same letter were different at 0.01 level of probability. c In postflowering LD alone (16 h) and night-break treatments of R and R → FR → R, the plants did not produce any new flowers and pods. At harvest, the plants kept vegetative status and did not show any flowering features nor reached any R stage.
after flowering was found to delay the senescence of leaves, and to cause resumption of vegetative growth of soybean, especially in photoperiod-sensitive varieties, besides its inhibitive effects on reproductive development. Exposure to LD could result in both floral reversion and inflorescence reversion after SD induction in soybean (Han et al., 1998; Washburn and Thomas, 2000). Han et al. (1998) proposed a new concept of whole plant reversion. The typical performance of the whole plant reversion was that the plants reverted to vegetative growth at many nodes, and not restricted to inflorescence or floral nodes. In this study, it was found that the resumption of vegetative growth after flowering was a direct LD effect, and not a secondary effect of abscission of flowers and pods. Even the SD-induced leaves produced before flowering could receive LD signals when plants were transferred to LD after flowering, indicating that soybean leaves could receive and respond to both SD and LD signals at various developmental stages. Such responses of plants depended on a signal-mediated mechanism involving the expression of growth promoters and inhibitors. The induced state of leaves was not permanent, but reversible when exposed to LD, even after floral commitment. Previous studies by Han and Wang (1995) proved that the responses of soybean to daylength after flowering are typical of photoperiodically controlled phenomena. Their results also showed that postflow-
ering photoperiod responses exist in various varieties belonging to different maturity groups (Han and Wang, 1996). The differences in the length of reproductive period among varieties with similar flowering dates and different maturity dates are positively correlated with the variation in postflowering photoperiod sensitivity (Han and Gai, 1998). Since the postflowering photoperiod treatments in the previous studies were imposed from beginning bloom, it was not fully proved if photoperiod was the major environmental factor controlling the development of soybean plants at middle and late reproductive phases, i.e., seed filling and maturation. Results from the present study showed that LD treatments at flowering, podding and seed filling stages all promoted the vegetative growth and inhibited reproductive development. In LD treatment, leaf senescence was delayed even when the LD treatment was imposed from full seed (R6), confirming the previous hypothesis (Han and Wang, 1995), which postulated that the photoperiod effects exist throughout the life cycle of the soybean plants. The presence of a phytochrome-mediated phenomenon is evidenced in the photoreaction that can be induced by exposure to red light and reversed by following far-red light exposure during the dark period (for review see Thomas and Vince-Prue, 1997). Parker et al. (1946) and Downs (1956) found that nightbreak with red light inhibits flowering of soybean. In the present study, it was found that effects of night-break after flowering also depend on whether R or FR is given
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as the final exposure in a series of R and FR light interruptions of the dark period. This phenomenon is similar to that reported for other Pfr-mediated responses (for review see Thomas and Vince-Prue, 1997). Final exposure to red light delayed or inhibited the reproductive development of Zigongdongdou, and the effects of red light could be reversed by exposure to far-red light. It is confirmed that the response of soybean to postflowering daylength is a typical phenomenon of photoperiodism. Based on previous (Han and Wang, 1995) and current results, it can be concluded that photoperiod responses of soybean exist from emergence to maturation, and phytochromes are the receptors of photoperiod signals throughout the whole life cycle of soybean. That is, soybean has the similar signal receiving mechanism before and after flowering. Leaves were proved to be the receivers of photoperiod signals that regulated reproductive development of soybean and other plants (for review see Thomas and Vince-Prue, 1997). However, it has not been well understood that if the reproductive organs play any roles in leaf growth and senescence, and other aspects of vegetative growth. Leopold et al. (1959) found that removal of the developing flowers and fruits delayed leaf senescence of plants. Caffaro and Nakayama (1988) proved that the removal of flowers affected the vegetative activity of the main stem terminal buds. Leopold et al. (1959) suggested that reproductive development might result in the diversion of mobilized reserve food materials towards the developing flowers and fruits. Sinclair and deWit (1975, 1976) proposed that the competitiveness of the seeds as sink abbreviates the period of seed filling by the onset of leaf senescence due to nitrogen stress. The results of the present study confirmed that pods (seeds) do promote the senescence of soybean leaves but also that photoperiod could regulate leaf senescence directly. It was proposed that, under inductive photoperiod conditions, leaves produce developmentpromoting substances including ABA (Brenner, 1987; Han and Gai, 1999), and result in the relocation of growth centers to reproductive organs in plants. The pods (seeds) could promote leaf senescence and maturation of whole plants, which may be exacerbated by nitrogen stress (Sinclair and deWit, 1975, 1976) and possibly by recycling of ABA back to leaves (Han and Gai, 1999). At the initial stage of the above pathway, SD signal received by leaves could be the triggering factor.
5. Conclusion Photoperiod is the major environmental factor regulating the postflowering development of soybean. The results in this paper showed that postflowering photoperiod not only controlled reproductive development of soybean, but also regulated leaf senescence, branching and other aspects of vegetative growth. Exposure to LD at flowering stage induced resumption of vegetative growth and abscission of flowers and pods in photoperiod-sensitive varieties. This phenomenon was previously referred to as ‘whole plant reversion’. Further studies in this paper indicated that the resumption of vegetative growth was the direct result of LD, instead of the secondary effects of abortion and abscission of reproductive organs. During the reversion process, the SD-treated leaves produced before flowering were the receiving organs of postflowering LD signals, showing that SD-treated leaves were functional under LD, and their physiological status was reversible. Results also proved that photoperiod effects on both vegetative growth and reproductive development persisted at all reproductive stages of soybean development, from the onset of flowering to seed filling and maturation. Night-break experiment demonstrated that postflowering photoperiod responses were R/FR reversible reactions and seemed to be mediated by phytochromes. The evidences obtained in the present studies further proved that the photoperiod responses were persistent throughout the whole life cycle of soybean.
Acknowledgements We thank Drs. Yonghua Yang, Yangnian Bai and Shikui Song for their comments on this manuscript; Dongxing Zhou, Chongbin Zhang, Shiwen Wang, Yuxi Shan, Haiqing Xu for plant care. We are grateful for detailed revision and helpful comments from two anonymous reviewers. This work was supported by the grants from the National Natural Science Foundation of China (30070456, 30070076), State Key Basic Research and Development Plan of China (2002CB111301), Beijing Natural Science Foundation (6012018), and the National High Technology Research and Development Program of China (2003AA207060-2).
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