Overexpression of E1 influences leaf development
To examine the function of E1 in other developmental pathways, we created four independent transgenic lines that express p35S:E1-Flag in DN50. Immunoblot analyses confirmed the expression of the recombinant E1 fusion protein in four independent T7 E1-OE lines (E1-OE1, E1-OE2, E1-OE3, and E1-OE4). The expression of the E1 protein line was highest in E1-OE4, followed by E1-OE3, E1-OE2, and E1-OE1 (Fig. 1a). Quantitative reverse transcription-PCR (qRT-PCR) analyses confirmed that the expression level of E1 was significantly higher in E1-OE lines than in DN50, and that E1 was constitutively and highly expressed in the E1-OE4 line, followed by E1-OE3, E1-OE2, and E1-OE1 (Fig. 1b).
As compared with DN50, E1-OE transgenic plants flowered significantly later under long-day conditions, and the plants were much shorter (Fig. S1a, 1b). Assessment of the unifoliolate leaves of E1-OE and DN50 plants at 7 DAE, revealed that E1-OE plants had smaller leaf areas and lower leaf weights than DN50 (Fig. 1c-e), they also curled downward (Fig. 1c). Higher E1 expression in the E1-OE lines was associated with increased curliness of the leaves (Fig. 1c).
We also observed the phenotypes of E1-OE transgenic lines in the W82 background. Consistent with our observations of E1-OE transgenics in DN50, leaf area and weight of E1-OE plants (E1-OE5-8) were smaller and lighter than those of W82 (Fig. S2a-c). Thus, E1 may regulate leaf development in soybean.
E1 regulates cell number and size in the developing leaf
Proper balance of leaf tissue structure is critical for normal leaf development . To further our understanding of the processes controlling leaf development, we analyzed transverse histological sections of E1-OE unifoliolate leaves. Compared with those in DN50 plants, cells were more disorganized in E1-OE plants (Fig. 2a). As an additional approach to examine the effects of E1-OE, we compared leaf functional traits such as leaf thickness, the cell tense ratio (CTR), spongy tissue ratio (SR), cell number and cell size (Fig. 2b-i). Leaf thickness was similar in E1-OE1 and E1-OE2 lines, but increased in E1-OE3 and E1-OE4 lines (Fig. 2b). The CTR, SR, and spongy tissue size were similar in E1-OE and DN50 plants (Fig. 2g, h, i). In contrast, E1-OE plants had significantly higher palisade tissue number, spongy tissue number and bulliform cell number (Fig. 2c-e), and lower palisade tissue size (Fig. 2f), confirming that the E1 could regulate leaf development by affecting the leaf tissue structure. We found that higher expression of E1 was associated with a more obvious cellular phenotype, confirming that E1 could influence the balance of different cells within the leaf tissue.
RNA-seq analysis of E1 overexpression
To identify the genes and signaling pathways related to E1-mediated leaf development, we performed RNA-seq analysis and analyzed the differentially expressed genes (DEGs) in the E1-OE transgenic and DN50 plants. The gene expression levels were similar between two biological replicates (Fig. 3a), but differed significantly between the E1-OE transgenic lines and DN50 plants. Genes involved in metabolic process, cellular process, single-organism process, response to stimulus, and biological regulation were enriched in the DEGs (Fig. 3b). We compared the RNA- seq datasets and identified a total of 7407 DEGs (FDR P < 0.01) (Supplmentary data 1). Among these, 3966 genes were significantly upregulated and 3441 genes were significantly downregulated (Fig. 3c). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that some primary metabolic pathways that are essential for plant growth and development were significantly enriched; these included fatty acid metabolism, phenylpropanoid biosynthesis, cysteine and methionine metabolism, and starch and sucrose metabolism (Fig. 3d).
RNA-seq approaches have identified transcription factor (TF) gene families, such as the AP2/ERF-ERF, bHLH, MYB, WRKY, NAC, HB-HD-ZIP, C2H2, GRAS, bZIP, MYB-related, TCP and B3-ARF families (Fig. 4a). We found 14 TCP TF genes among the DEGs, of which six were CIN-type TCP genes (Fig. 4a). The heat map showed that 5 CIN-type TCP (TCP6, TCP14, TCP15, TCP30, and TCP33) genes were significantly downregulated in E1-OE transgenic lines as compared with DN50 (Fig. 4b).
E1 represses TCP genes
The soybean genome includes 19 CIN-subclass TCP genes (Feng et al., 2018). To validate the RNA-seq results and E1 regulation in all 19 CIN-type TCP genes in soybean, we tested their expression by qRT-PCR in E1-OE and DN50 plants. Most genes were downregulated in E1-OE transgenic plants, including TCP6, TCP7, TCP11, TCP13, TCP14, TCP18, TCP19, TCP29, TCP47, and TCP49 (Fig. 4c-n). However, the expression levels of TCP15, TCP36, TCP39, and TCP42 remained unchanged (Fig. S3b, d, e), and TCP32, TCP33 and TCP37 were upregulated in E1-OE transgenics (Fig. S3a, c). The expression levels of TCP30 and TCP38 were not detected in E1-OE and DN50 plants.
To examine the regulatory effect of E1 on its target genes, we performed transient expression assays, using TCP14 and TCP29 promoters fused to the LUC reporter (pTCP14:LUC and pTCP29:LUC; Fig. 5a). The effector construct harbored E1 under the control of the 35S promoter (p35S:E1; Fig. 5a). We transformed the reporter construct (pTCP14:LUC or pTCP29:LUC) and the effector construct (p35S:E1) into healthy N. benthamiana leaves and found that E1 significantly repressed TCP14 and TCP29 expression (Fig. 5b). Thus, E1 regulates leaf development by repressing CIN-type TCPs.
To determine whether E1 directly inhibits the expression of TCP genes, we performed a ChIP-qPCR assay to compare the relative enrichment of specific TCP14 and TCP29 sequences in E1-OE and DN50 plants using anti-Flag antibodies. We selected four sites in the 2027 bp and 2209 bp regions upstream of the TCP14 and TCP29 promoters, respectively (Fig. 5c). The E1 protein was highly enriched in the TCP14 promoter sites 1 and 4, and in the TCP29 promoter site 1, whereas it was present at extremely low levels in the DN50 control (Fig. 5c). These results showed that E1 could directly bound the promoters of TCP genes.
The transcript levels of the CIN-type TCP genes in soybean tissues
To understand the functions of CIN-type TCP genes in soybean, we use an RNA-seq database and retrieved transcript levels of 10 of the TCP genes repressed by E1, in eight different tissues (flower, leaf, pod, shoot, nodule, cotyledon, seed and root; Machado et al., 2020). These genes presented similar expression profiles and were constitutively expressed to high levels in the leaf and cotyledon (Fig. 6a-k). In contrast, all CIN-type TCP genes displayed low transcript abundance in nodule and root, except TCP6 high expression level in seed and root (Fig. 6a-k). Moreover, TCP13, TCP47, and TCP49 presented similar expression profiles and were highly expressed in pod, flower and shoot (Fig. 6b, e, j, k). TCP7, TCP14, TCP19, TCP11, TCP18, and TCP29 were expressed in shoot and seed, seed, flower and shoot, shoot, pod and shoot, and flower, respectively (Fig. 6c, f, h, d, g, i).
To determine the tissue-specific expression patterns of TCP genes, we assayed the transcript levels of 10 CIN-type TCP genes by qRT-PCR. The tissue-specific expression patterns in the qRT-PCR were similar to those in the RNA-seq data (Fig. 6l). Thus, the CIN-type TCP genes regulated by E1 play key roles in soybean leaf development.