A GmRAV ortholog is involved in photoperiod and sucrose control of flowering time in soybean

Abstract

Photoperiod and sucrose levels play a key role in the control of flowering. GmRAV reflected a diurnal rhythm with the highest expression at 4 h after the beginning of a dark period in soybean leaves, and was highly up-regulated under short-day (SD) conditions, despite of not following a diurnal pattern under long-day (LD) conditions. GmRAV-i (GmRAV-inhibition) transgenic soybean exhibited early flowering phenotype. Two of the FT Arabidopsis homologs, GmFT2a and GmFT5a, were highly expressed in the leaves of soybeans with inhibition (-i) of GmRAV under SD conditions. Moreover, the transcript levels of the two FT homologs in GmRAV-i soybeans were more sensitive to SD conditions than LD conditions compared to the WT plant. GmRAV-i soybeans and Arabidopsis rav mutants showed more sensitive hypocotyl elongation responses when compared with wild-type seedlings, and GmRAV-ox overevpressed in tobacco revealed no sensitive changes in hypocotyl length. These indicated that GmRAV was a novel negative regulator of SD-mediated flowering and hypocotyl elongation. Although sucrose has been suggested to promote flowering induction in many plant species, high concentration of sucrose (4% [w/v]) applied into media defer flowering time in Arabidopsis wild-type and rav mutant. This delayed flowering stage might be caused by reduction of LEAFY expression. Furthermore, Arabidopsis rav mutants and GmRAV-i soybean plants were less sensitive to sucrose by the inhibition assays of hypocotyls and roots growth. In contrast, transgenic GmRAV overexpressing (-ox) tobacco plants displayed more sensitivity to sucrose. In conclusion, GmRAV was inferred to have a fundamental function in photoperiod, darkness, and sucrose signaling responses to regulate plant development and flowering induction.

Figure 1. Quantitative real-time RT-PCR analysis of transcript level of GmRAV gene under SDs and LDs.

A, Tissue-specific expression of soybean GmRAV in SDs. Tissues tested are leaf (Tl), trifoliate leaf (TL), stem (St), root (RO), pod (PO), flower bud (FB), and immature seed (IS) (plants aged 21 d). B, Relative transcript levels of GmRAV mRNA in soybean leaves under SDs and LDs. Soybean leaves were harvested every 4 h for 48 h at 25-day-old under LDs and SDs. Open and closed boxes indicate days and nights. C, Time course-dependent expression in LDs. Soybean ‘Dong Nong 47’ plants were grown under LDs for 10 d and were transferred to LDs or SDs before sampling. Relative transcript levels were analyzed by qRT-PCR. D, Histochemical detection of GmRAV–GUS promoter activity in transgenic Arabidopsis seedlings. 4-day-old seedlings were grown on MS medium.

Figure 2. A, Phenotypes of the T6 generation GmRAV-i soybean under LDs and SDs.

50-day-old seedlings of WT and GmRAV-i transgenic soybean under LDs and SDs at Harbin (planted at May 28). B, The compare of the number of flower buds between WT plants and GmRAV-i soybeans under LDs and SDs. The flower buds of 50 plants were measured for each treatment. Error bars represent the SE. **Significant differences in comparison to the non-transgenic lines at P<0.01 (Student’s t test). C and D, Diurnal expression of soybean FT homologs: GmFT2a and GmFT5a in GmRAV-i soybeans grown under SDs (8 h/16 h light/dark). Trifoliate leaves were sampled every 4 h at 15 DAE. White and black bars at the top represent light and dark phases, respectively. Samples were processed and analyzed by RT-PCR as described in Experimental procedures. The levels of GmACTIN expression were used as a normalization control, respectively. Average and SE values for three replications are given for each data point. E and F, Relative transcript levels of GmFT2a and GmFT5a mRNA in GmRAV-i soybean leaves under SDs and LDs. Soybean leaves were harvested at 4 h before dawn at 25-day-old under LDs and SDs. G, Pathway controlling flowering in response to short days in soybean.

Figure 3. Effects of day length on hypocotyl length in 9-day-old wild-type and GmRAV-i soybean seedlings under LDs and SDs.

A, Histograms of the mean (n = 20) for seedlings grown on medium. All seedlings were transgenic for the soybeans indicated. The seedlings were scored 9 d after sowing. Scale bar = 10 mm. **Significant differences in comparison to the non-transgenic lines at P<0.01 (Student’s t test). B, Representative seedlings are shown.

Figure 4. Effects of day length on hypocotyl length in 9-day-old wild-type and Arabidopsis rav mutants under LDs and SDs.

A, Histograms of the mean (n = 20) for seedlings grown on medium. The seedlings were scored 9 d after sowing. Scale bar = 10 mm. *differences in comparison to the non-transgenic lines at 0.01<P<0.05, **Significant differences in comparison to the non-transgenic lines at P<0.01 (Student’s t test). B, Phenotype of 9-day-old WT seedlings and Arabidopsis rav mutants on MS medium under LDs and SDs. C, Representative seedlings are shown.

Figure 5. Effects of sucrose on flowering time for Arabidopsis.

A, Arabidopsis rav mutants and WT seedlings were grown on media with various concentrations of sucrose for 2 weeks, and then transferred to soil under LDs. The flowering time of seedlings with 2% sucrose was WT (24±0.5) and Arabidopsis rav mutant (21±0.8). WT plants are the control for Arabidopsis rav mutants. Values are the average of 30 to 45 plants. The error bars indicate one SE of the mean. Similar results were obtained in two independent experiments. *Differences in comparison to the non-transgenic lines at 0.01<P<0.05, **Significant differences in comparison to the non-transgenic lines at P<0.01 (Student’s t test). B, Phenotypes of the Arabidopsis rav mutant. 23-day old seedlings of WT and Arabidopsis rav mutant under natural day length (LD) with treated by 2% and 4% sucrose. C, Quantitative real-time RT–PCR analysis of LFY expression in Arabidopsis rav mutants. Control amplification of 18 s rRNA transcript indicated equal amounts of cDNA.

Figure 6. Response of wild-type and GmRAV-ox tobacco seedling to sucrose.

A and B, Relative hypocotyl and root growth in response to various concentrations of sucrose. The length of hypocotyl of tobacco seedlings grown 2% (w/v) sucrose was 4.91±0.75 mm for the WT and 5.85±0.76 mm for GmRAV-ox. Root length, of seedlings grown 2% (w/v) sucrose was 15.8±1.21 mm in the WT and 18.8±1.94 mm in GmRAV-ox. The hypocotyl and root length of 20–30 seedlings were measured for each treatment. Error bars represent the SE. C, Phenotype of 7-day-old WT seedlings and T3 generation GmRAV-ox tobaccos on MS medium containing 2% and 4% sucrose. *Differences in comparison to the non-transgenic lines at 0.01<P<0.05 (Student’s t test).

Figure 7. Response of WT and Arabidopsis rav mutants to sucrose on development.

A and B, Relative hypocotyl and root growth in response to various concentrations of sucrose. Hypocotyl length, as a percentage of the untreated control, of seedlings grown on sucrose. The length of hypocotyl of Arabidopsis seedlings grown 2% (w/v) sucrose was 4.26±0.24 mm for the WT and 4.06±0.46 mm for Arabidopsis rav mutant. Root length, as a percentage of the untreated control, of seedlings grown on sucrose. Root length, of seedlings grown without sucrose was 26.70±1.16 mm in the WT and 28.24±1.33 mm in Arabidopsis rav mutant. The hypocotyl and root length of 20–30 seedlings were measured for each treatment. Error bars represent the SE. C, Phenotype of 7-day-old WT seedlings and Arabidopsis rav mutants on MS medium containing 2% and 4% sucrose.

Figure 8. Response of WT and GmRAV-i soybean seedlings to sucrose.

A and B, Relative hypocotyl and root growth in response to various concentrations of sucrose. Hypocotyl length, as a percentage of the untreated control, of seedlings grown on sucrose. The length of hypocotyl of soybean seedlings grown without sucrose or glucose was 57.17±1.30 mm for the WT and 61.17±1.97 mm for GmRAV-i soybean. Root length, as a percentage of the untreated control, of seedlings grown on sucrose. Root length, of seedlings grown without sucrose or glucose was 60.33±1.30 mm in the WT and 65.67±1.97 mm in GmRAV-i. Error bars represent the SE. C, Relative number of lateral roots in response to various concentrations of sucrose. The number of lateral roots, as a percentage of the untreated control, of seedlings grown on sucrose. Error bars represent the SE. The number of lateral roots of seedlings grown without sucrose and glucose was 17.83±0.75 for the WT and 21.17±0.98 for GmRAV-i soybean. The number of lateral roots of 20–30 seedlings was scored for each treatment. *Differences in comparison to the non-transgenic lines at 0.01<P<0.05. D, Phenotypes of 7-day-old WT and GmRAV-i soybean seedlings on MS medium containing 2% and 4% sucrose. *Differences in comparison to the non-transgenic lines at 0.01<P<0.05 (Student’s t test).

Figure 9. Response of the WT plants, Arabidopsis rav mutants and GmRAV-ox tobaccos or GmRAV-i soybean seedlings to full strength MS media supplemented with the indicated sugar concentration.

A and B, Relative root length in response to 200-day-old Arabidopsis and tobaccos. Root length, as a percentage of the untreated control, of seedlings grown on carbon source. Error bars represent the SE. Root length, of seedlings grown without carbon sucrose was WT (15.96±1.2 mm) and Arabidopsis rav mutant (19.25±0.8 mm), and for tobaccos without carbon sucrose was WT (15.29±0.9 mm) and GmRAV-ox (18.4±1.2 mm). Root length of 20–30 seedlings was measured for each treatment. C and D, Relative hypocotyl and root length in response to 200 mM carbon source in 8-day-old soybeans. Hypocotyl and root length, as a percentage of the untreated control, of seedlings grown on carbon source. Error bars represent the SE. Hypocotyl length without carbon source was WT (50.67±1.2 mm) and GmRAV-i (54.83±0.9 mm), and main root length was WT (67±1.2 mm) and GmRAV-i (79.83±0.7 mm). E, Relative number of lateral roots in response to 200 mM carbon source. Number of lateral roots, as a percentage of the untreated control, of seedlings grown on carbon source. Error bars represent the SE. The number of lateral roots, of seedlings grown without carbon source was WT (16.33±0.5) and GmRAV-i (21±1.0). Number of lateral roots of 20–30 seedlings was scored for each treatment. *differences in comparison to the non-transgenic lines at 0.01<P<0.05, **Significant differences in comparison to the non-transgenic lines at P<0.01 (Student’s t test).