Overexpression of <i>ZmSPL12</i> confers enhanced lodging resistance through transcriptional regulation of <i>D1</i> in maize

Abstract

Introduction of the semi-dwarf genes (sd1 in rice, RhtB1b and RhtD1b in wheat) has drastically increased lodging resistance and grain yields in these two important crops since the 1960s, resulting in the so called ‘Green Revolution’ (Hedden, 2003). Maize (Zea mays L.) is one of the most widely cultivated cereal crops in the world and increasing planting density is an effective strategy to increase grain yield (Duvick, 2005). However, high planting density could trigger shade avoidance response and cause increased plant height and ear height, prone to lodging and yield loss. Thus, reducing plant/ear height and increasing lodging resistance is a continuous goal for maize breeders. ‘Green Revolution’ genes in rice or wheat are both either involved in GA biosynthesis or signalling. Previous studies have identified a number of maize dwarf mutants that are defective in GA biosynthesis or signalling, such as anther ear1, dwarf plant1 (d1), dwarf3, dwarf8 and dwarf9 (Lawit et al., 2010; Teng et al., 2013). However, these mutants could not be used in breeding because of pleiotropic detrimental effects, such as dwarfism, small ear size, and yield losses. Thus, identification of semi-dwarf genes is desirable for genetic improvement of lodging resistance in maize. D1, encoding a GA 3-oxidase (ZmGA3ox2) catalysing the final step of bioactive GA synthesis, is a candidate gene for qPH3.1, a major QTL for plant height in maize (Teng et al., 2013). To identify candidate transcription factors (TFs) that negatively regulate D1 expression in maize, we first performed WGCNA co-expression network analysis of D1 using the published transcriptomic data (Stelpflug et al., 2016). We initially identified 67 TFs showing negative expression correlation with that of D1 in the shoot apical meristem (SAM) and internodes (cut-off value ≤ −0.8). Among them, we found 10 candidate TFs that potentially bind to the D1 promoter (~3 kb upstream of the translation initiation site) using plantpan 3.0 (Chow et al., 2019), including ZmARF18 (Zm00001d014377), ZmSPL12 (Zm00001d015410), ZmGN1(Zm00001d007842), ZmbZIP104 (Zm00001d015421), ZmMYBR77 (Zm00001d036632), ZmbHLH128 (Zm00001d054038), ZmNAC123 (Zm00001d035084), ZmMADS73 (Zm00001d018142), ZmbHLH55 (Zm00001d012067) and ZmbHLH117 (Zm00001d024783) (Figure 1a). We next selected top six TFs with the highest correlation, including ZmARF18, ZmSPL12, ZmGN1, ZmbZIP104, ZmMYBR77 and ZmbHLH128, and tested their binding to D1 promoter. Yeast one-hybrid assay showed that only ZmSPL12 could bind to D1 promoter (Figure 1b). Transient expression assay showed that ZmSPL12 strongly repressed the expression of the pD1::LUC reporter gene (Figure 1c). Quantitative reverse transcription PCR (RT-qPCR) assay revealed that ZmSPL12 was mainly expressed in seedling at V2 stage, SAM and internodes at the V8 and V10 stages (Figure 1d). Subcellular localization assay revealed that ZmSPL12 protein was exclusively localized to the nucleus (Figure 1e). To investigate the role of ZmSPL12, we generated Zmspl12 knockout plants using the CRISPR/Cas9 technology. Two independent lines (ko#1 and ko#2) with frame-shift mutations were selected and used for phenotypic investigation. Both ko#1 and ko#2 plants exhibited significantly higher plant height and ear height (Figure 1f). While two independent transgenic ZmSPL12 overexpression lines (ZmSPL12-OE, #499 and #500) showed reduced plant height and ear height, compared with the non-transgenic control plants, and that the degree of reduction in plant height is negatively correlated with the expression levels of ZmSPL12 (Figure 1g). As expected, the expression level of D1 was decreased significantly in the ZmSPL12-OE plants (Figure 1h). Histological observation revealed that internode cells in the ZmSPL12-OE plants were significantly shorter than those of control plants (Figure 1i), indicating that the shorter internode in the ZmSPL12-OE plants is mainly caused by decreased cell length. Moreover, measurement of stalk strength showed that the ZmSPL12-OE plants were significantly stronger than that of the control plants (Figure 1j). To confirm the effect of ZmSPL12 on plant height is caused by altered GA levels, we measured endogenous bioactive GA levels, including GA1, GA3, GA4 and GA7 in the internodes. The results showed that the levels of these GAs were all significantly decreased in the ZmSPL12-OE plants (Figure 1k). In addition, treatment of the ZmSPL12-OE plants with exogenous GA3 effectively restored plant height (Figure 1l). These results support the notion that ZmSPL12 acts upstream of D1 to regulate GA biosynthesis, thus plant height and ear height. To test the potential utility of ZmSPL12 in maize breeding, we planted ZmSPL12-OE (#500) and #500-CK under three different planting densities (45 000, 90 000, and 135 000 plants per hectare) in Hainan, China in 2020. The ZmSPL12-OE plants had significantly reduced plant height, ear height and more grain yield than the #500-CK plants under all three planting densities (Figure 1m,n). Moreover, the stalk strength of ZmSPL12-OE plants enhanced significantly compared with the non-transgenic plants under high planting densities (90 000 and 135 000 plants per hectare; Figure 1o). Further, we introgressed the ZmSPL12-OE#500 transgene into two elite parental inbred lines, Chang7-2 and PH6WC, through repeated backcrossing. The plant height and ear height of both improved Chang7-2#500 and PH6WC#500 reduced significantly, compared with their respective original inbred lines (Figure 1p,q). Together, our results demonstrate that ZmSPL12 could mimic the function of ‘Green Revolution’ genes to confer reduced plant height and increased lodging resistance, thus facilitating high-density planting and increased yield in maize. This research was supported by grants from the National Key Research and Development Program of China (2021YFF1000301, 2020YFE0202300), the National Natural Science Foundation of China (32022065) and Hainan Yazhou Bay Seed Lab (B21HJUS01). The authors declare no conflict of interests. HW and BW conceived and designed the project. BZ conducted the experiments. YZ, YL, HX, DK, YX and ZZ participated in some experiments. CL analysed the data. BZ and MX wrote the manuscript. HW revised the manuscript.