Overexpression of <i>GmWRI1b</i> in soybean stably improves plant architecture and associated yield parameters, and increases total seed oil production under field conditions

Abstract

The functions of WRINKLED1 (WRI1) transcription factor in regulating fatty acid (FA) biosynthesis are highly conserved in crop plants, including maize (Zea mays; Pouvreau et al., 2011), rapeseed (Brassica napus; Li et al., 2015), oil palm (Elasis guineensis; Ma et al., 2013), camelina (Camelina sativa; An et al., 2017) and soybean (Glycine max; Chen et al., 2018; Chen et al., 2020; Zhang et al., 2017). Recently, Kong et al. (2017) found that the WRI1 plays a role in root auxin homeostasis and affects root development in Arabidopsis, suggesting its involvement in root architecture and potentially shoot architecture as well. In soybean, there are two WRI1 homologs (GmWRI1a and GmWRI1b), and the expression levels of GmWRI1a showed a positive correlation with the seed oil content (SOC; Zhang et al., 2017), whereas such correlation between GmWRI1b and SOC remains unknown. The ‘RY(CATGCA)’ cis-element, to which the GmABI3a (an ortholog of Arabidopsis ABSCISIC ACID INSENSITIVE3) protein directly binds, is absent in the GmWRI1b promoter, resulting in low GmWRI1b expression levels and consequently only basal function of the GmWRI1b in soybean (Zhang et al., 2017). Chen et al. (2018) reported that overexpression of GmWRI1a increased the SOC and FA content, and altered the FA composition in soybean. However, the detailed functions of GmWRI1b in soybean remain elusive. Recently, individual overexpression of GmWRI1a or GmWRI1b gene in soybean hairy roots altered phospholipid and galactolipid syntheses, soluble sugar and starch contents in nodules derived from transgenic hairy roots, and exhibited an increase in nodule number (Chen et al., 2020). On the other hand, knockdown of both GmWRI1a and GmWRI1b genes in soybean hairy roots interfered with glycolysis and lipid biosynthesis in developed nodules, and resulted in a decrease in nodule number (Chen et al., 2020). Although Chen et al. (2020) explored the overexpression of the GmWRI1 genes in hairy roots for functional studies, their results suggest that the GmWRI1 genes may have other pleiotropic functions in addition to regulation of genes related to FA synthesis in soybean. Furthermore, the previous work only looked at the hairy root system; and therefore, it could not monitor the phenotypic outcomes at whole-plant level (Chen et al., 2020). Here, we obtained three stable transgenic soybean lines overexpressing GmWRI1b (GmWRI1b-OX) (Figure 1a), and examined the agronomic traits of the T4-generation homozygous GmWRI1b-OX plants grown under field conditions at three types of interval distances (10, 30 and 40 cm) between two plants within a row in the Hanchuan Transgenic Biosafety Station in 2019. All three GmWRI1b-OX lines showed improved agronomic performance (Figure 1b), including decreases in plant height (at 10- and 30-cm plant distances) and internode length (Figure 1c–d), but increases in node number, branch number, stem diameter, shoot dry weight, pod number per plant, seed number per plant, yield per plant and yield per ha at three plant density levels, when compared with wild-type (WT) plants (Figure 1e–l). In addition, the SOCs were found to be higher in GmWRI1b-OX than in WT plants at all three types of plant distances (Figure 1m). As a result, the total seed oil production per plant increased by 41.3% to 54.8% at 40-cm, 39.3% to 60.2% at 30-cm, and 63.8% to 93.2% at 10-cm distances (Figure 1n). The seed protein contents and 100-seed weights of three GmWRI1b-OX lines and WT were found to be comparable (Figure 1o, p). These data collectively indicate that GmWRI1b is a promising gene, which can be used to alter plant architecture, thereby improving yield, and increase SOC in soybean and perhaps in other crops. Previously, Chen et al. (2018) reported that overexpression of GmWRI1a also increased yield per plant, which was resulted from the larger seed size, not from the changes in node number, pod number per plant and seed number per plant. In the present study, overexpression of GmWRI1b in soybean resulted in the increase in yield per plant in GmWRI1b-OX lines (Figure 1k), which was the result of improvement of plant architecture, including the increase in the pod number per plant (Figure 1i) and seed number per plant (Figure 1 j), but not seed size (Figure 1p). This finding was not reported by any previous study on any GmWRI1 genes. It would be then interesting to investigate whether the GmWRI1a also plays a role in the regulation of soybean architecture. It should also be mentioned that in this study, overexpression of GmWRI1b elevated SOC but did not impact total protein content (Figure 1m and o). Recently, Manan et al. (2017) reported that ectopic expression of the G. max LEAFY COTYlEDON2a (GmLEC2a), which has a role in up-regulating the expression of GmWRI1 in soybean, increased not only the SOC but also the protein content in transgenic Arabidopsis seeds. Thus, the relationship between protein and oil contents in soybean deserves detailed investigations on a case-by-case basis. Plant architecture is one of the important factors for the development of high-yield cultivars. Previously, ectopic expression of the Arabidopsis CYP714A2 in rice caused semi-dwarfism with moderately decreased plant height, more yielding tillers and higher grain yield in comparison with WT plants, which was due to gibberellic acid (GA) deactivation (Zhang et al., 2011). We found that the expression levels of a soybean CYP714A gene (GmCYP714A/Glyma.18G218500) significantly increased in the OX6 and OX8 plants (Figure 1q). Previous studies have reported that the GmWRI1s bind to the AW-box ‘CnTnG(n)7CG’ in the promoters of genes in soybean (Chen et al., 2020; Chen et al., 2018). We next employed electrophoretic mobility shift assay (EMSA) and transactivation assay to validate whether GmWRI1b would directly regulate the expression of GmCYP714A. Results of these assays convincingly indicated that the GmWRI1b could directly bind to the AW-box motif identified in the GmCYP714A promoter (Figure 1r), and activate its expression (Figure 1s). We also found that the endogenous GA3 and GA4 levels were significantly reduced in the GmWRI1b-OX6 line, while the GA1 and GA7 levels were comparable in OX6 and WT plants (Figure 1t). The semi-dwarf phenotype of GmWRI1b-OX6 plants was rescued by GA3 application (Figure 1u), suggesting the involvement of GmWRI1b in GA catabolism, probably through regulating the expression of its downstream gene GmCYP714A. These results demonstrated that overexpression of GmWRI1b improved plant architecture and yield per plant in soybean under field conditions by altering GA metabolism. Our findings also provide a strategy and opportunity for stably increasing yield and SOC in soybean using a single gene. The research was supported by the National Genetically Modified Organisms Breeding Major Projects (2016ZX08004-003 and 2016ZX08004-005). The funding body had no role in the design of the study and collection, analysis and interpretation of data and in writing the manuscript. W Guo, L-SP Tran, XA Zhou and D Cao conceived and designed the experiments. W Guo, LM Chen, HL Yang, HF Chen, QB You, AL Bao, SL Chen, QN Hao, Y Huang, DZ Qiu, ZH Shan, ZL Yang, SL Yuan, CJ Zhang and XJ Zhang performed the experiments and analysed the data. YQ Jiao generated the transgenic soybean plants. L-SP Tran, W Guo and D Cao wrote the paper. The authors declare that they have no competing interests. Not applicable. Not applicable.