Plant Biotechnology Journal ( IF 13.8 ) Pub Date : 2020-01-07 , DOI: 10.1111/pbi.13324 Wei Guo 1 , Limiao Chen 1 , Haifeng Chen 1 , Hongli Yang 1 , Qingbo You 1 , Aili Bao 1 , Shuilian Chen 1 , Qingnan Hao 1 , Yi Huang 1 , Dezhen Qiu 1 , Zhihui Shan 1 , Zhonglu Yang 1 , Songli Yuan 1 , Chanjuan Zhang 1 , Xiaojuan Zhang 1 , Yongqing Jiao 1, 2 , Lam-Son Phan Tran 3, 4 , Xinan Zhou 1 , Dong Cao 1
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.
中文翻译:
大豆中GmWRI1b的过表达稳定地改善了植物结构和相关的产量参数,并增加了田间条件下的种子油总产量。
WRINKLED1(WRI1)转录因子在调节脂肪酸(FA)生物合成中的功能在包括玉米(Zea mays ; Pouvreau et al。,2011),油菜籽(Brassica napus ; Li et al。,2015)在内的农作物中高度保守。,油棕(Elasis guineensis ; Ma等,2013),茶花(Camelina sativa ; An等,2017)和大豆(Glycine max ; Chen等,2018 ; Chen等,2020 ;张)等。,2017)。最近,Kong等。(2017)发现WRI1在根生长素稳态中起作用,并影响拟南芥的根发育,表明其参与根构型以及潜在的芽构型。在大豆中,有两个WRI1同源物(GmWRI1a和GmWRI1b),并且表达水平GmWRI1a显示出与种子油含量正相关(SOC;张等人,2017),而之间的这种相关性GmWRI1b和SOC仍然不明。“ RY(CATGCA)”顺式GmWRI1b启动子中不存在GmABI3a(拟南芥ABSCISIC ACID INSENSITIVE3的直系同源物)蛋白直接结合的元素,导致GmWRI1b的表达水平低,因此大豆中GmWRI1b的基础功能低(Zhang等,2017)。)。Chen等。(2018)报道,GmWRI1a的过表达增加了大豆的SOC和FA含量,并改变了大豆的FA组成。但是,GmWRI1b在大豆中的详细功能仍然难以捉摸。最近,GmWRI1a或GmWRI1b的个别过表达大豆毛状根中的基因改变了磷脂和半乳糖脂的合成,转基因毛状根中的根瘤中可溶性糖和淀粉含量改变,并且根瘤数增加(Chen et al。,2020)。另一方面,大豆毛状根中的GmWRI1a和GmWRI1b基因的敲低干扰了发达结节中的糖酵解和脂质生物合成,并导致结节数减少(Chen等人,2020年)。虽然陈等。(2020)探索了毛状根中GmWRI1基因的过表达,进行了功能研究。除了调控与大豆中FA合成相关的基因外,GmWRI1基因还可能具有其他多效性功能。此外,以前的工作仅着眼于毛状根系统。因此,它不能监测全植物水平的表型结果(Chen等,2020)。
在这里,我们获得了3种过表达GmWRI1b(GmWRI1b‐OX)的稳定转基因大豆品系(图1a),并研究了田间条件下以三种间隔距离(10、30和10)在田间生长的T4代纯合GmWRI1b‐OX植物的农艺性状。并在2019年的汉川转基因生物安全站中的两株植物之间间隔40厘米(约40厘米)。所有三个GmWRI1b‐OX品系显示改良的农艺性能(图1b),包括降低株高(在10和30厘米的植株距离)和节间长度(图1c–d),但节数,枝数,茎直径,茎干增加与野生型(WT)植物相比,重量,单株荚数,单株种子数,单株产量和每公顷产量在三种植物密度水平下(图1e-1)。此外,在所有三种植物距离上,GmWRI1b-OX的SOC均比野生型植物高(图1m)。结果,每株植物的总籽油产量在40 cm处增长了41.3%,达到54.8%,在30 cm处增长了39.3%,达到60.2%,在10 cm距离增长了63.8%,达到93.2%(图1n)。三种GmWRI1b‐OX的种子蛋白含量和100种子重量发现品系和野生型具有可比性(图1o,p)。这些数据共同表明,GmWRI1b是一个很有前途的基因,可用于改变植物结构,从而提高产量,并增加大豆以及其他作物的SOC。
以前,Chen等。(2018)报道GmWRI1a的过表达也增加了每株植物的产量,这是由于种子更大,而不是节点数,单株荚数和每株种子数的变化引起的。在本研究中,大豆中GmWRI1b的过表达导致GmWRI1b - OX系的单株产量增加(图1k),这是植物结构改善的结果,包括单株荚数增加(图1i) )和每株种子的数量(图1 j),而不是种子大小(图1p)。以前任何有关GmWRI1的研究均未报告这一发现。基因。然后,研究GmWRI1a在大豆结构调节中是否也发挥作用将是很有趣的。还应该提到的是,在这项研究中,GmWRI1b的过表达提高了SOC,但并未影响总蛋白含量(图1m和o)。最近,Manan等人。(2017)报道G.max LEAFY COTYlEDON2a(GmLEC2a)的异位表达在上调大豆中GmWRI1的表达中起作用,不仅增加了SOC,而且增加了转基因拟南芥中的蛋白质含量种子。因此,应根据具体情况对大豆中蛋白质与油含量之间的关系进行详细研究。
植物结构是发展高产品种的重要因素之一。以前,与野生型植物相比,拟南芥CYP714A2在水稻中的异位表达导致半矮化,株高适中降低,分decreased更高,籽粒产量更高,这是由于赤霉素(GA)失活所致(Zhang et al。,2011)。)。我们发现大豆CYP714A基因(GmCYP714A / Glyma.18G218500)的表达水平在OX6和OX8植物中显着增加(图1q)。先前的研究已报道GmWRI1s与大豆基因启动子中的AW-box'CnTnG(n)7CG'结合(Chen等,2020 ; Chen等。,2018)。接下来,我们采用电泳迁移率迁移分析(EMSA)和反式激活分析来验证GmWRI1b是否会直接调节GmCYP714A的表达。这些分析的结果令人信服地表明,GmWRI1b可以直接与GmCYP714A启动子中鉴定的AW-box基序结合(图1r),并激活其表达(图1s)。我们还发现,GmWRI1b - OX6品系的内源性GA 3和GA 4水平显着降低,而GA 1和GA 7在OX6和WT植物中的水平相当(图1t)。GA 3的应用挽救了GmWRI1b - OX6植物的半矮表型(图1u),表明GmWRI1b参与了GA分解代谢,可能是通过调节其下游基因GmCYP714A的表达来实现的。这些结果表明,在田间条件下,通过改变GA代谢,GmWRI1b的过表达改善了大豆的植物结构和单株产量。我们的发现也为使用单一基因稳定提高大豆产量和SOC提供了策略和机会。