About the Authors:
Guangyu Guo
Contributed equally to this work with: Guangyu Guo, Kun Xu, Xiaomei Zhang
Affiliation: College of Agriculture, Northeast Agricultural University, Harbin, China
Kun Xu
Contributed equally to this work with: Guangyu Guo, Kun Xu, Xiaomei Zhang
Affiliations MOA Key Lab of Soybean Biology (Beijing), National Key Facility of Crop Gene Resource and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie, Haidian District, Beijing, China, Graduate School of Chinese Academy of Agricultural Sciences, Beijing, China
Xiaomei Zhang
Contributed equally to this work with: Guangyu Guo, Kun Xu, Xiaomei Zhang
Affiliation: MOA Key Lab of Soybean Biology (Beijing), National Key Facility of Crop Gene Resource and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie, Haidian District, Beijing, China
Jinlong Zhu
Affiliation: MOA Key Lab of Soybean Biology (Beijing), National Key Facility of Crop Gene Resource and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie, Haidian District, Beijing, China
Mingyang Lu
Affiliations MOA Key Lab of Soybean Biology (Beijing), National Key Facility of Crop Gene Resource and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie, Haidian District, Beijing, China, Graduate School of Chinese Academy of Agricultural Sciences, Beijing, China
Fulu Chen
Affiliation: MOA Key Lab of Soybean Biology (Beijing), National Key Facility of Crop Gene Resource and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie, Haidian District, Beijing, China
Linpo Liu
Affiliation: MOA Key Lab of Soybean Biology (Beijing), National Key Facility of Crop Gene Resource and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie, Haidian District, Beijing, China
Zhang-Ying Xi
Affiliation: College of Agronomy, Henan Agricultural University, Zhengzhou, China
Andreas Bachmair
Affiliation: Dept. of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Dr. Bohr Gasse 9, A-1030 Vienna, Austria
Qingshan Chen
* E-mail: [email protected] (Y-FF); [email protected] (QC)
Affiliation: College of Agriculture, Northeast Agricultural University, Harbin, China
Yong-Fu Fu
* E-mail: [email protected] (Y-FF); [email protected] (QC)
Affiliation: MOA Key Lab of Soybean Biology (Beijing), National Key Facility of Crop Gene Resource and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie, Haidian District, Beijing, China
Introduction
Photoperiod sensitivity is an important trait, which allows crops to adapt to diverse latitudinal environments for flowering and maturation at a suitable season. In plants, many genes have been identified until now that are involved in the photoperiodic pathway of flowering. Two key genes, CONSTANS (CO) and FLOWERING LOCUS T (FT), form a central regulon (CO-FT) for flowering regulation [1]. The CO-FT regulon is a highly conserved module in many plants including Arabidopsis and crops, with diversification across a diverse groups of angiosperms [2, 3]. The regulon contributes to ecogenetic variation in a highly adaptive trait in different plants [4].
Even though FT, belonging to PEBP (Phosphatidyl Ethanolamine-Binding Protein) gene family [5, 6], is a highly conserved florigen across plants, FT and its like-genes in different versions may perform distinct functions in different plants [7]. For example, Arabidopsis has a main gene, FT, which controls photoperiodic flowering [8], while it homeolog TSF participates in flowering regulation in a special pathway that mediates cytokinin input [9]. AtFT and AtTSF are also involved in regulation of lateral shoot outgrowth [10]. In rice, two FT homologs, Hd3a and RFT1, control flowering in short day and long day conditions, respectively [11–13]. Maize has an FT-like family, including ZCN8, which is a good candidate of florigen [14]. The potato floral and tuberization transitions are controlled by two different FT-like paralogs (StSP3D and StSP6A) that respond to independent environmental cues [15]. Two FTs in Populus contribute flower initiation, but FT1 functions in winter, while FT2 in summer [16]. In tomato, FT ortholog SFT not only accelerates flowering in a dose-dependent manner, but also enhances fruit set [17, 18]. The BvFT1 and BvFT2 paralogs in beet act antagonistically on flowering regulation and growth habit [19]. FT also regulates Arabidopsis stomatal opening in a cell-autonomous mode [20], flower development [21] and inflorescence meristem stabilization [22]. The soybean genome contains at least six FT homeologs (GmFTL1-6), which show flowering activity in Arabidopsis [23–25]. Two of them, GmFTL3 (a.k.a GmFT2a) and 4 (a.k.a. GmFT5a) (S1 Table) were experimentally confirmed to enhance flowering in soybean [26–28].
CO is a zinc finger transcriptional factor of the B-box family (BBX) family [29] and also conserved in the plant kingdom including algae [30–32]. During the evolutional history, CO family genes diverged with diverse functions but kept the photoperiodic-/circadian-regulated characters: Chlamydomonas growth [31], Arabidopsis [29] and rice [33] flowering, potato tuberization [34], the short-day-induced growth cessation and bud set occurring in the fall in aspen [4]. However, it is proven that not all CO-like genes regulate FT expression, and divergence of functions is widely spread across plants [3].
Soybean, originating from South China (lower latitude region), is a typical short-day plant for flowering induction. After genetic divergence, soybean underwent domestication (artificial selection) 6.000–9.000 year ago resulting in many cultivars, which adapted different environmental conditions in different areas [35]. In the soybean domestication, maturity is one of the important agronomic traits and closely related to flowering time. During the long history of soybean domestication, the sensitivity of soybean to the length of light irradiation was weakened. Thus, many soybean cultivars can flower in higher-latitude (northern, with long duration of sunshine) regions [36, 37]. For example, Kennong 18, a cultivar from Harbin (in northern of China, located at EL125°42′-130°10′ and NL44°04′-46°40′) can flower even under long-day photoperiod (18 hrs/6 hrs, light/dark) [38], indicating that northern soybean cultivars are much less sensitive to photoperiods and their critical-day-lengths are longer than that of their southern ancestors. The locus affecting the time of maturity in soybean was firstly designated as E locus by Owen [39]. Until now, there are at least ten loci (nine E and one J loci), which are reported to function in flowering and maturation in soybean: E1 and E2 [40], E3 [41], E4 [42], E5 [43], E6 [44], E7 [45], E8 [46], E9 [47] and J [48]. E1 encodes a protein containing a putative bipartite nuclear localization signal and a region distantly related to B3 domain [49], E2 is a Gigantea (GI) gene [50], E3 [51] and E4 [52] are phytochrome A genes. These loci regulate flowering time through GmFTLs.
In this study, the detailed expression spectra of FT-like and CO-like genes under field conditions were analyzed to evaluate the expression divergences of different gene copies among different cultivars. In addition, both ectopic overexpression and silencing of GmFTL homologs in soybean were also employed to confirm GmFTL functions in photoperiodic flowering. The results show that all GmFTLs contribute to flowering control and GmCOLs regulate GmFTLs’ expression. Additionally, GmFTLs exhibit additional functions beyond flowering regulation.
Results
Northern soybean cultivars have much longer critical-day-length
To understand the relationship between soybean florigen gene GmFTLs and flowering habits in field conditions, we employed three northern groups (two cultivars for each) with different maturities (early, middle, and late) in local fields (Table 1). All cultivars show semi-determinate growth habits except Beidou 5, which is indeterminate. During the growth season, the local durations of sunshine ranges from 16 to 18 hours (Fig 1), which are typical long-day conditions for most of plants including soybean. It is well known that Glycine max cv Biloxi, a typical soybean cultivar for photoperiod study in the last century, has a critical day length of 13.5 hours [53]. Therefore, these data confirmed that the cultivars employed here were all photoperiod-hyposensitive. To compare their flowering response to day length, six oybean cultivars were grown in a Harbin region, where the day length was about 16.5 hours during the growth season and shorter than that of their original locations (Fig 1), to make sure all cultivars can flower and mature normally.The results showed that early cultivars had three fully-opened trifoliolates when flowering, whereas middle and late ones had four trifoliolates. Then, to investigate potential differences of different leaves in flowering induction, we harvested individual leaves according to developmental stages for real time quantitative PCR (RT-qPCR) analysis of genes, including GmFTLs, GmCOLs, and E genes.
[Figure omitted. See PDF.]
Fig 1. The day length at origin locations of different soybean cultivars during growth season.
Day length data are from the Era Shuttle Calendar (http://if.ustc.edu.cn/~ygwu/calendar.html). All cultivars were planted on May 11 for sampling at different dates (bold letters on X-axis) at Harbin district, Heilongjian province, China.
https://doi.org/10.1371/journal.pone.0136601.g001
[Figure omitted. See PDF.]
Table 1. The information of cultivars used in this study.
https://doi.org/10.1371/journal.pone.0136601.t001
Expression of E genes in different soybean cultivars
To analyze the activity of E1, E2, E3 and E4 in soybean cultivars used in this study, we employed RT-qPCR to evaluate their expression in short day conditions. The results show that E1, E2, E3, E4 and E2 homeologs (GmGI1 and GmGI2, [54]) expressed both in the morning (ZT8),and in the evening (ZT20) in all cultivars to different extents (Fig 2). Therefore, these E genes may not mutate and work normally in these cultivars.
[Figure omitted. See PDF.]
Fig 2. E gene expression levels in leaves of seven soybean cultivars.
Relative expression levels (REL) were analyzed by RT-qPCR and normalized to UKN1. Averages and standard errors are the result of three replicates. For detailed information on stages and sampling, see Materials and Methods section.
https://doi.org/10.1371/journal.pone.0136601.g002
GmFTL3 is most abundant among GmFTLs regardless of cultivars
To understand the functional divergence of GmFTLs in flowering control, we carried out extensive analysis of expression patterns in different cultivars with different maturities (Table 1). The total transcripts of different GmFTLs in soybean increased along with plant growth, and reached peaks at the stage of the 3rd trifoliolates (T3 stage, Fig 3). Among them, GmFTL3 exhibited both more or less identical patterns in all cultivars and the highest abundance among the FT homologs in all stages, suggesting that GmFTL3 may play a most important role for flower induction in different cultivars. The expression of GmFTL5, the closest homeolog of GmFTL3 (a pair-wise homeolog, with close relationship; [23]), did not show such a pattern among cultivars, indicating its expression divergences among cultivars. However, the other two pair-wise homeologs (GmFTL1 and GmFTL2 or GmFTL4 and GmFTL6) displayed similar changes across plant growth in a given cultivar, even though some differences existed among different cultivars. Fig 3 also showed the phenomenon that early maturity cultivars (Heihe 35 and Beidou 51) had relatively higher levels of GmFTLs. The results imply that all GmFTLs might contribute to flowering induction in soybean cultivars, and higher levels of GmFTLs probably lead to early flowering and rapid maturity.
[Figure omitted. See PDF.]
Fig 3. Relative expression levels of GmFTLs in six soybean cultivars at different stages.
U, U-stage; T1, T1-stage; T2, T2-stage; T3, T3-stage; T4, T4-stage. Relative expression levels (REL) were analyzed by RT-qPCR and normalized to UKN1. Averages and standard errors are the result of three replicates. For detailed information on stages and sampling, see Materials and Methods section.
https://doi.org/10.1371/journal.pone.0136601.g003
All individual leaves express GmFTLs
Next, we analyzed the contribution of different leaves to production of GmFTLs for flowering control. Therefore, we analyzed the patterns of all GmFTLs in different leaves (Fig 4). As expected, the expression of a given GmFTL was not restricted to specific leaves, but detectable in all leaves. However, the abundance of different GmFTLs varied in different leaves and also had no consistency among cultivars. For example, in Heihe 35, unifoliolates and the first and third trifoliolates had relatively high level of GmFTLs, while the second trifoliolates produced less GmFTLs; but for Beidou 51, it appeared to be the opposite, especially when plants were flowering (T3 stage). The results indicate that all leaves contribute to flowering control, but to different extents in a cultivar-dependent mode.
[Figure omitted. See PDF.]
Fig 4. Relative expression level of GmFTLs in different leaves in cultivars Heihe 35 and Beidou 51 at different stages.
Leaves: U, unifoliolate; T1, the 1st trifoliolate; T2, the 2nd trifoliolate; T3, the 3rd trifoliolate stage. Relative expression levels (REL) were analyzed by RT-qPCR and normalized to UKN1. Averages and standard errors are the result of three replicates. For detailed information on stages and sampling, see Materials and Methods section.
https://doi.org/10.1371/journal.pone.0136601.g004
GmFTLs show a developmentally regulated expression
In early maturity cultivars (Heihe 35 and Beidou 51), expression of different GmFTLs in various leaves appeared to increase with developmental progress (Fig 3). In comparison with expression levels in a given leaf in early maturity cultivars, three pair-wise homeologous GmFTL genes (GmFTL3 vs. GmFTL5, GmFTL1 vs. GmFTL2, or GmFTL4 vs. GmFTL6) exhibited expression divergence, especially after plants emerged flowers. In case of middle (Heihe 45 and Beidou 5, S1 Fig) and late (Kenfeng 16 and Hefeng 43, S2 Fig) maturity cultivars, there were similar patterns, even though the relationship between GmFTL expression and both flowering time and maturity are less stringent than that in early maturity cultivars.
GmFTLs exhibit a divergence of circadian expression
Circadian expression is a typical character of florigen gene FT. A given GmFTL expressed in similar diurnal patterns, even though different patterns prevailed among different GmFTLs (Fig 5), indicating robust, but different circadian patterns. Pair-wise GmFTLs (GmFTL1 and GmFTL2, GmFTL4 and GmFTL6) also showed similar diurnal patterns, whereas the patterns of GmFTL3 and GmFTL5 were quite divergent (Fig 5, S3 and S4 Figs). In Arabidopsis, the FT expressing peak occurs at dusk [55]. In early stages, there were two peaks of FT transcripts, one in the morning (at 8:00), and the other in the afternoon (at 16:00), respectively (Fig 5, S3 and S4 Figs). The results show that different diurnal-regulation mechanisms exist for FT homeologs between soybean and Arabidopsis.
[Figure omitted. See PDF.]
Fig 5. The diurnal rhythm of GmFTL expression in cultivars Heihe 35 and Beidou 51 at different stages.
Relative expression levels (REL) were analyzed by RT-qPCR and normalized to UKN1. Averages and standard errors are the result of three replicates. For detailed information on stages and sampling, see Materials and Methods section.
https://doi.org/10.1371/journal.pone.0136601.g005
GmCOL1, GmCOL2, GmCOL5, and GmCOL13 may all contribute to GmFTL expression
In various plants, the FT gene is regulated by multiple genes, and CONSTANS (CO) is the main up-regulator of FT in the photoperiodic pathway [1, 7]. Soybean also has multiple copies of CO homologs [23]. So the question is that which one of the CO-like genes regulates a specific FT homolog for flowering in soybean. To evaluate it, we further investigated the expression pattern of GmCOLs with the same set of samples used for GmFTL expression. Based on our previous studies [23], we focused on four CO homolog candidates (GmCOL1 and GmCOL2, GmCOL5, and GmCOL13) in soybean cultivars Heihe 35 and Beidou 51. As expected, GmCOL1 and GmCOL2 or GmCOL5 and GmCOL13 had patterns similar to each other in developmental (Fig 6), tissue-/organ- (Fig 7), and circadian (Fig 8) regulation as the GmFTLs. The level of GmCOL1 and GmCOL2 were obviously higher than that of GmCOL5 and GmCOL13. In case of the circadian patterns, GmCOL1 and GmCOL2 peaked in the morning, while the highest level of GmCOL5 and GmCOL13 appeared in the afternoon. This characteristic mirrored the two peaks of GmFTLs, respectively (Fig 5). Comparison of GmCOL expression among different cultivars showed similar modes (S5–S8 Figs). The results suggest that individual GmCOLs do not regulate one specific GmFTL, but morning GmCOL1/2 may be related to the morning peak of GmFTLs, while GmCOL5/13 contribute the afternoon peak of GmFTLs.
[Figure omitted. See PDF.]
Fig 6. Relative expression level analyses of GmCOLs in six soybean cultivars at different stages.
Relative expression levels (REL) were analyzed by RT-qPCR and normalized to UKN1. Averages and standard errors are the result of three replicates. For detailed information on stages and sampling, see Materials and Methods section.
https://doi.org/10.1371/journal.pone.0136601.g006
[Figure omitted. See PDF.]
Fig 7. Relative expression level analyses of GmCOLs in different leaves in cultivars Heihe 35 and Beidou 51 at different stages.
Relative expression levels (REL) were analyzed by RT-qPCR and normalized to UKN1. Averages and standard errors are the result of three replicates. For detailed information on stages and sampling, see Materials and Methods section.
https://doi.org/10.1371/journal.pone.0136601.g007
[Figure omitted. See PDF.]
Fig 8. The diurnal rhythm of GmCOL expression in cultivars Heihe 35 and Beidou 51 at different stages.
Relative expression levels (REL) were analyzed by RT-qPCR and normalized to UKN1. Averages and standard errors are the result of three replicates. For detailed information on stages and sampling, see Materials and Methods section.
https://doi.org/10.1371/journal.pone.0136601.g008
Ectopic expression of GmFTL4 results in early flowering in soybean
We expressed GmFTL4-GFP driven by the 35S promoter in cultivar Tianlong 1, a cultivar from central China (location: EL113°41′- 115°05′, NL28°58′- 31°22′). We got 20 independent transgenic lines in total. Most of the lines (62%) showed early flowering phenotypes with the shortest flowering time of 21 days after germination (Fig 9C) in the Zhengzhou area (location: EL112°42′- 114°14′, NL34°16′- 34°58′) (S9 Fig). The first flowers appeared in unifoliolate axils in transgenic lines (Fig 9B), except one line (#16–3), where they appeared in the first, third or fourth trifoliolate axils. In comparison, flowers in the parent line appeared in the third or fourth axils (Fig 9A). Four lines were selected for further analysis of gene expression. Data from RT-qPCR (Fig 9D) clearly displayed that transgenic GmFTL4 over-expressed in the lines, and their levels were corresponding to flowering phenotypes, that is, the higher the level, the earlier flowering (Fig 9D and 9E). We then investigated the effect of exogenous GmFTL4 on the expression of endogenous GmFTLs. The results showed the fluctuation of different GmFTLs’ expression was observed to various extents compared to that in WT soybean (Fig 9G–9L): in three lines (#16–2, #16–3, and #19–1) the expression of GmFTL1, 2, 4, 5, and 6 was dampened, while in line #16–1 the expression of these genes was enhanced. The change of GmFTL3 expression was interesting: enhanced in line #16–3, repressed in other lines. In our opinion, these effects may possibly result from different insertion positions of T-DNA and/or interfering/co-suppression between exogenous and endogenous GmFTL genes. The phenotype of flowering times was a complex result of interaction between exogenous and endogenous genes. Another obvious and interesting character was the relationship between GmFTL3 and the position of the first flowers. Higher GmFTL3 (Line #16–3) resulted in flowers appearing in trifoliolate axils and lower GmFTL3 (Line #16–1, #16–2, and #19–1) resulted in flowers appearing in unifoliolate axils, indicating a role of GmFTL3 in shoot growth.
[Figure omitted. See PDF.]
Fig 9. Overexpression of GmFTL4 causes precocious flowering in the soybean cultivar Tianlong 1.
(A) A wild-type Tianlong 1 plant showing the first flower appearing at the 3rd trifoliolate axil (red arrow-heads). (B) A transgenic line indicating flowers (red arrow-heads) appearing at the unifoliate (yellow words) and the first trifoliolate axil (red arrow and words indicating the petiole of the first trifoliolate). (C) The flowering times of different transgenic lines compared with wild-type plants (WT). (D) Relative expression level (REL) analyses of transgene-derived GmFTL4 in transgenic lines and wild-type plants (WT). The total number of leaves at flowering and flowering times of the transgenic plants were shown in (E) and (F), respectively. (G) to (L) Relative expression level analyses of endogenous GmFTLs in transgenic lines in field conditions. Trifoliolates were sampled at 40 days after sowing.
https://doi.org/10.1371/journal.pone.0136601.g009
Knock-down of GmFTL expression delays flowering in soybean
To further investigate the function of GmFTLs, RNA silencing approach was employed. Due to highly conserved sequences among GmFTLs, an RNAi-fragment based on GmFTL1 coding-sequence (cds) was used as the target sequence, which was expected to target all GmFTLs (S10A Fig). Sixteen independent lines were generated. Most lines displayed late flowering phenotype with the latest one of 50 days after sowing compared with 38 days for WT (Fig 10A and 10B). We grouped these lines according to their flowering times in 2 day intervals (Fig 10C), and selected nine lines from different groups to check the silencing efficiency. RT-qPCR data indicated that all endogenous GmFTLs were knocked down in all lines with the largest effect on GmFTL3 (95.6%, S10B Fig). The efficiency of knock-down was also consistent with the extent of flowering time delay (Fig 10C–10I and S10B Fig), suggesting that all of GmFTLs may contribute to flowering control. However, the relative level of GmFTL3 was still higher than that of other GmFTLs (Fig 10D–10I), and these transgenic lines had no flowers in unifoliolate axils, further inferring that GmFTL3 functioned in determination of the flower position on the stem.
[Figure omitted. See PDF.]
Fig 10. Silencing GmFTLs delays flowering and inhibits growth.
(A) A GmFTL-RNAi transgenic (right) and wild type (left) soybean plants grown in the field for 40 days. (B) Flowering time of transgenic lines. The total number of plants: WT, 69; Transgenic lines, 643. (C) The flowering time of transgenic lines used for analysis of GmFTL relative expression level (REL, D to I). (D to I) Endogenous GmFTL relative expression level in GmFTL silencing lines. Trifoliolates were sampled at 40 days after sowing and leaves from five plants of each line were mixed for RT-qPCR (C to I). Data are mean +/- SD from three technical replicates.
https://doi.org/10.1371/journal.pone.0136601.g010
Another significant phenotype was that all GmFTL-RNAi plants had shorter/smaller stature than WT (Fig 10A). Generally, knock-down or knock-out of flowering enhancers result in a higher/larger stature phenotype in Arabidopsis [55] and rice [13]. Therefore, the dwarf phenotype inferred additional and different functions of GmFTLs in soybean.
Discussion
Accumulating evidences support that FT, a highly conserved florigen gene across plants, occurs in many plants in multiple copies, which display functional variation [7, 56]. In soybean, there are at least six GmFTLs showing flowering activity [23, 24, 26–28]. However, the functional divergence of GmFTLs that occurred in soybean cultivars is unknown. In this study, we focus on analysis of GmFTL expression spectra in different cultivars under field conditions, and show that GmFTLs’ expression correlates with cultivar maturity, in that early maturity cultivars have higher levels of GmFTLs. And knockdown of GmFTLs delay flowering. Such a conclusion is in accordance with what happens in other species, including Arabidopsis, rice, wheat (review in [1, 57–59]). This suggests that all of GmFTLs impact on flowering induction and domestication-related diversification of soybean. In comparison with expression levels in a given leaf in early maturity cultivars, three pair-wise homeologous GmFTL genes (GmFTL3 vs. GmFTL5, GmFTL1 vs. GmFTL2, or GmFTL4 vs. GmFTL6) exhibit expression divergence, especially after plants emerge flowers. In case of diurnal expressions, different GmFTLs also show various patterns, even the patterns of GmFTL3 and GmFTL5 (pair-wise genes) are quite divergent. Such expression differentiation may contribute to survival of duplicated genes [60] after genome duplication events during a long-term evolutional progress [61, 62]. The expression pattern of GmFTL3 in different soybean cultivars is quite similar and highly conserved, but other GmFTLs have cultivar-specific patterns with relatively low levels, indicating that with progress of domestication, soybean GmFTL3 retained the conserved function in flowering regulation, while GmFTL1/2/4/5/6 underwent divergence to adapt to different environments.
Even though different copies of GmFTLs display different expression levels, GmFTL3 (GmFT2a) shows the highest abundance among them, regardless of developmental-, organ-, and circadian-specific expressions and cultivars, indicating that GmFTL3 may be the major florigen, that is consistent with previous report [24, 26]. However, the function of other GmFTLs on the regulation of flowering time can’t be ignored, because exogenous GmFTLs expressed from the 35S promoter enhance flowering in soybean or Arabidopsis (this study, [23, 24, 26–28], and the pattern of developmental and circadian expressions is still similar among GmFTLs. To analyze flowering phenotype of GmFTL mutants and to express GmFTLs from their native promoters in soybean will help to understand their contribution to florigen.
GmFTL3 not only has high abundance, but also a unique circadian pattern,with two peaks in the early stage, one in morning and the other one in the afternoon. Other GmFTLs have their unique peak expressions in the morning or afternoon. On the other way, the transcript level of both GmCOL1/2 peaks in the morning, while that of GmCOL5/13 peaks in the afternoon. We speculate that there may be at least two CO-FT regulons in soybean flowering network, one includes GmCOL1/2 and morning-phase GmFTLs, the other contains GmCOL5/13 and afternoon-/evening-phase GmFTLs. These two CO-FT regulons may coordinately control flowering time in soybean. Certainly, more lines of evidence are needed to support this speculation.
Many studies in Arabidopsis used whole seedlings, which include many leaves and roots, as materials for investigation of flowering induction. However, there is no evidence showing a differential influence of different leaves on flowering regulation, even though most of Arabidopsis wild types have at least 10 leaves before flowering. Recent evidences show that cotyledons alone are enough for Arabidopsis floral induction [63]. In Pharbitis nil [64] and pea [65], cotyledons are also involved in flower initiation. Soybean plants produce several (3–4 for cultivars used here) trifoliolate leaves before the first flower emerges. However, our previous research indicated that unifoliolates are sufficient to induce soybean flowering [38]. To sum up our data of expression analysis, all leaves express different GmFTLs. Therefore, all leaves may contribute to floral induction.
Previous papers reported that maturity loci E1, E2, E3 and E4 have important, but different impacts on maturity and photoperiod response, and they also indicate that it is unique in photoperiod sensitivity for different cultivars and there are other genes involved in photoperiod response [66–68]. In this study, we did not found parallel relationship between E1 expression and both GmFTL/GmCOL expression and cultivars maturity for these six soybean cultivars. E1 abundance of transcripts displays an opposite relationship to cultivar maturity except cultivars Heihe 35 and Tianlong 1, similar to previous reports [67, 69]. A similar situation was found for E2, E3 and E4. This information may indicate much complicated mechanisms of GmFTL regulation happened in different cultivars.
Expression data also show that GmFTL3 is the gene expressed at the highest level gene in different cultivars, and the first flowers appears in the trifoliolate axils of WT plants. Overexpression of GmFTLs results in formation of flowers in unifoliolate axils (this study and [26]). In overexpressing transgenic line #16–3, there are no flowers in unifoliolate axils even though exogenous GmFTL4 is overexpressed. Detailed comparison of expression levels of GmFTLs in different transgenic lines reveals that the expression balance between GmFTL3 and other GmFTLs in plants contributes to the position of the first flowers.
GmFTLs may also participate the regulation of plant vegetative growth. In Arabidopsis [55] and rice [13], FT expression enhances flowering and inhibits vegetative growth. Therefore, knock-down or mutation of the FT gene results in late flowering and higher and larger stature of plants. However, all knock-down transgenic lines of soybean in this study have much shorter stature than their parents, suggesting that GmFTLs are involved in stem growth in soybean. An alternative explanation is that GmFTLs may control both reproduction and vegetative growth in a GmFTL-species dependent mode.
Introduction of elite soybean varieties into different latitude areas is important but challenging, because soybean is a qualitative/absolute short-day plant [70], whose flowering is highly sensitivity to day length. Therefore, photoperiod sensitivity is a key factor, which limits elite introduction between different areas. We show that overexpression or silencing of GmFTLs can significantly change flowering time. Therefore, it is possible to design soybean plants with a desired flowering time by controlling the expression level of GmFTLs. For instance, plant breeders can introduce transgenic lines with special flowering times, as shown in this study, into a soybean elite variety of interest to modulate its flowering time. Of course, the effect of modulation of GmFTL expression on other agronomic traits, such as yield and resistance to biotic/abiotic stress, should be carefully evaluated.
Materials and Methods
Plant Materials
Six soybean cultivars are from the northeastern area, Heilongjian province of China as Table 1 showed. All seeds from Dr. Qingshan Chen lab at College of Agriculture, Northeast Agricultural University. These cultivars were grown in pots for sampling, or in the same field with the same cultivation management for phenotype assessment and seed reproduction in Harbin, Heilongjiang Province, China (Fig 1). For sampling, six seeds from one pot were sown on May 11, 2013, and harvested on Aug.18, 2013 (for Heihe 35 and Beidou 51), or on Sept. 13, 2013 (for Heihe 45, Beidou 5, Kenfeng 16 and Hefeng 43). The samples of leaves for RT-qPCR analysis were harvested on July, 1/7/12/18, 2013, respectively. For phenotype and seed reproduction, the seeds of all cultivars were sown on Apr. 25, 2013, and harvested on Sept. 20, 2013. The unifoliolate and different trifoliolate leaves were sampled at different stages before flowering. Stages were defined as the time when the unifoliolates or trifoliolates expanded fully, therefore we harvested samples at unifoliolate stage (U-stage), the 1st trifoliolate stage (T1-stage), the 2nd trifoliolate stage (T2-stage), the 3rd trifoliolate stage (T3-stage), and the 4th trifoliolate stage (T4-stage). For developmental samples, different leaves were combined at a given stage. For circadian samples, leaves were collected at 4 hour intervals at different stages (S2 Table). All the samples above were combined with at least five individual plants. For transgenic plants, five middle-leaflets of the 3rd trifoliolates (counting from the top) from five individual plants were combined as samples at day 40 after sowing. Three groups of samples from different pods were harvested for the parallel analysis. All samples were immediately frozen in liquid nitrogen and stored at -80°C until used.
Transgenic plants were grown in the field of Zhengzhou, China, which locates at EL112°42'- 114°14' and NL34°16'- 34°58', and is little higher latitude than Wuhan, China (EL113°41′- 115°05′, NL28°58′- 31°22′), a city where its WT cultivar Tianlong 1 originated from. The daylength change during the growth season is shown in S9 Fig The seeds were sown on June 10, 2014, and harvested on Oct 12, 2014. The samples of leaves for RT-qPCR analysis were harvested on July 18, 2014.
For expression evaluation of E genes, 7 cultivars were grown in a growth chamber under short day conditions (8 hrs/16 hrs, light and dark; light intensity: 270 μmol·m-1·s-1). Leaf samples of the 1st trifoliolates at the 1st trifoliolate stage (T1-stage) were harvested at Zeitgeber (ZT) 8 and ZT20 for RT-qPCR analysis.
RNA isolation, cDNA synthesis and RT-qPCR analysis
Total RNA was extracted from various leaves using TRIzol (Invitrogen, USA). The RNA was treated with RNase-free recombinant DNase I (Takara, Dalian, China). The integrity of the RNA was checked electrophoretically and quality assessment of total RNA was checked with NanoDrop ND-2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The isolated RNA was then subjected to reverse transcription using the Super-Script III Reverse Transcriptase kit. Real-time quantitative PCR (RT-qPCR) was performed with the SYBR Green Mix (Takara, Dalian, China) on StepOnePlus Real-Time PCR System (Applied Biosystems, USA) according to the manufacturer’s protocol. GmUKN1 (Glyma12g02310) was selected as the reference gene. Three independent biological replicates were performed. Raw data were standardized as described previously [71]. All primers are listed in S3 Table.
Construction of plasmids
The full-length CDS of GmFTL4 was amplified from our previous clone [23] using the primer pairs of GmFTL4-Asc I-5941-F and GmFTL4-Xba I-R (S3 Table), and then inserted into the pFGC5941-GFP vector, which was constructed by inserting the GFP gene into pFGC5941 (GenBank Accession No AY310901) between Xba I and Sma I sites, to obtain pFGC5941-GmFTL4-GFP as GmFTL4-GFP expressing construct. For GmFTLs silencing, a fragment of GmFTL1 (S10 Fig) was cloned into pFGC5941 at Nco I / Asc I and Avr II / Xba I sites, to obtain binary vector pFGC5941-GmFTL. Therefore, both GmFTL4 and GmFTL1-silencing fragments were driven by the cauliflower mosaic virus 35S promoter.
Soybean genetic transformation
The Agrobacterium strain EHA105 containing constructs pFGC5941-GmFTL4-GFP or pB7G-GmFTL was used to transform the soybean cultivar Tianlong 1, following the cotyledonary node method [72–74]. Transformants were screened by applying160 mg/L glufosinate onto the preliminary leaves of the seedlings. Transgenic plants in the greenhouse and field were sprayed with 1/1000 herbicide Basta. The herbicide-resistant plants were subjected to molecular and phenotypic analysis. For phenotype (flowering and stature) analysis, T2 and T3 lines (for RNAi transgenic plants) or T1 (for overexpressing transgenic plants) were used.
Supporting Information
[Figure omitted. See PDF.]
S1 Dataset.
https://doi.org/10.1371/journal.pone.0136601.s001
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S1 Fig. Tissue-specific expression patterns of GmFTLs in soybean cultivars HeiHe 45 and BeiDou 5.
https://doi.org/10.1371/journal.pone.0136601.s002
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S2 Fig. Tissue-specific expression patterns of GmFTLs in soybean cultivars Kenfeng 16 and Hefeng 43.
https://doi.org/10.1371/journal.pone.0136601.s003
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S3 Fig. Circadian expression patterns of GmFTLs in soybean cultivars HeiHe 45 and BeiDou 5.
https://doi.org/10.1371/journal.pone.0136601.s004
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S4 Fig. Circadian expression patterns of GmFTLs in soybean cultivars Kenfeng 16 and Hefeng 43.
https://doi.org/10.1371/journal.pone.0136601.s005
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S5 Fig. Tissue-specific expression patterns of GmCOLs in soybean cultivars HeiHe 45 and BeiDou 5.
https://doi.org/10.1371/journal.pone.0136601.s006
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S6 Fig. Tissue-specific expression patterns of GmCOLs in soybean cultivars Kenfeng 16 and Hefeng 43.
https://doi.org/10.1371/journal.pone.0136601.s007
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S7 Fig. Circadian expression patterns of GmCOLs in soybean cultivars HeiHe 45 and BeiDou 5.
https://doi.org/10.1371/journal.pone.0136601.s008
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S8 Fig. Circadian expression patterns of GmCOLs in soybean cultivars Kenfeng 16 and Hefeng 43.
https://doi.org/10.1371/journal.pone.0136601.s009
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S9 Fig. The day length change in Zhengzhou.
https://doi.org/10.1371/journal.pone.0136601.s010
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S10 Fig. The strategy and efficiency of GmFTL silencing.
https://doi.org/10.1371/journal.pone.0136601.s011
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S1 Table. The names of GmFTLs in this study and literature.
https://doi.org/10.1371/journal.pone.0136601.s012
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S2 Table. Sampling organs and date/time for gene expression analysis of circadian.
https://doi.org/10.1371/journal.pone.0136601.s013
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S3 Table. The list of primers used in this study.
https://doi.org/10.1371/journal.pone.0136601.s014
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Acknowledgments
Ethics Statement: Northeast Agricultural University and Henan Agricultural University issued the permission for fields in Harbin and Zhengzhou, respectively.
Author Contributions
Conceived and designed the experiments: Y-FF QC. Performed the experiments: GG KX XZ JZ ML FC LL ZX. Analyzed the data: GG KX XZ Y-FF. Contributed reagents/materials/analysis tools: Y-FF QC. Wrote the paper: Y-FF GG KX AB.
Citation: Guo G, Xu K, Zhang X, Zhu J, Lu M, Chen F, et al. (2015) Extensive Analysis of GmFTL and GmCOL Expression in Northern Soybean Cultivars in Field Conditions. PLoS ONE 10(9): e0136601. https://doi.org/10.1371/journal.pone.0136601
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Abstract
The FLOWERING LOCUS T (FT) gene is a highly conserved florigen gene among flowering plants. Soybean genome encodes six homologs of FT, which display flowering activity in Arabidopsis thaliana. However, their contributions to flowering time in different soybean cultivars, especially in field conditions, are unclear. We employed six soybean cultivars with different maturities to extensively investigate expression patterns of GmFTLs (Glycine max FT-like) and GmCOLs (Glycine max CO-like) in the field conditions. The results show that GmFTL3 is an FT homolog with the highest transcript abundance in soybean, but other GmFTLs may also contribute to flower induction with different extents, because they have more or less similar expression patterns in developmental-, leaf-, and circadian-specific modes. And four GmCOL genes (GmCOL1/2/5/13) may confer to the expression of GmFTL genes. Artificial manipulation of GmFTL expression by transgenic strategy (overexpression and RNAi) results in a distinct change in soybean flowering time, indicating that GmFTLs not only impact on the control of flowering time, but have potential applications in the manipulation of photoperiodic adaptation in soybean. Additionally, transgenic plants show that GmFTLs play a role in formation of the first flowers and in vegetative growth.
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Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer