TABLE CAPTIONS and FIGURE LEGENDS
Table 1 Differentially expressed genes (DEGs) related to FA motabolism in the WT and NtMYB12a-OE tobacco leaves. DEGs with FDR < 0.05 and |log2 ratios|≥1.00 are listed here.
Table 2 The DEGs induced by sucrose treatment andNtMYB12a over expression.
FIGURE 1 The phylogenetic tree and sequence analysis of six NtMYBs. (a) The selected R2R3-MYB transcription factors was classified into SG5, SG6, and SG7 subgroups, which was associated with the biosynthesis of PA, anthocyanin, and flavonol, respectively. (b) Alignment of the six NtMYB protein sequences. The amino acids identical in six sequences, five sequences, and four sequences are highlighted in black, dark gray, and light gray, respectively. The R2R3-type MYB domains are indicated above the alignment, while the SG7 and SG7-2 motifs are highlighted by red boxes. (c) Exon/intron structure of the six NtMYB genes. Black boxes represent exons, and lines represent introns. Numbers indicate length of the exons and introns. The triangle indicates the PAM positon for Crispr/cas9.
FIGURE 2 Expression pattern of the tobacco NtMYB genes. (a) Relative expression levels of six NtMYB genes in various tobacco tissues. (b) Relative expression levels of NtMYB12a andNtMYB12b genes in tobacco leaves from different development stages. Values are means of three independent replicates. Error bars denote standard deviations. (c) Subcellular localization of NtMYB12a-GFP and NtMYB12b-GFP fusion proteins. NLS is a marker protein for nuclear localization; Merge, merging of NLS, GFP, Chloroplast autofluorescence and Brightfield images.
FIGURE 3 Functional differentiation of NtMYB12a and NtMYB12b in regulating the biosynthesis of flavonoids in tobacco. (a) Gene-specific fragment used for the construction of RNAi vectors. (b) Relative expression levels of the NtMYB12a and NtMYB12b genes in different positive RNAi lines. Asterisks represent statistically significant differences between WT and RNAi plants determined by two-tailed paired Student’s t-test (**, P < 0.01; *, P < 0.05). (c) Flavonol content in the tobacco leaves of different RNAi lines. (d) Anthocyanin content in the tobacco leaves of different RNAi lines. Values are means of three independent replicates. Error bars denote standard deviations. The significant differences among different lines are detected by Tucky’s test.
FIGURE 4 Quantitative assay of the flavonoids content in the tobacco leaves and flowers. (a) Inflorescence phenotypes of the WT,NtMYB12a-OE and ntmyb12a mutant plants. Bar=1cm. Anthocyanin content in the flowers (b) and leaves (c) of WT,NtMYB12a-OE and ntmyb12a mutant plants. (d) Flavonol content in the flowers and leaves of WT, NtMYB12a-OE andntmyb12a mutant plants. Asterisks represent statistically significant differences between WT and transgenic plants determined by two-tailed paired Student’s t-test (**, P < 0.01; *, P < 0.05).
FIGURE 5 NtMYB12a repressed the accumulation of FA in tobacco leaves. (a &b) Relative expression levels of the FA-related DEGs in the leaves of WT, NtMYB12a-OE , NtMYB12a-RNAi , andntmyb12a mutant plants. Total FA content (c) and percentage of PUFAs (d) in the leaves of WT, NtMYB12a-OE , NtMYB12a-RNAi , and ntmyb12a mutant plants. Asterisks represent statistically significant differences between WT and transgenic plants determined by two-tailed paired Student’s t-test (**, P < 0.01; *, P < 0.05).
FIGURE 6 Comparison of the gene expression levels and FA content in tobacco seeds. (a) Relative expression levels of the eight FA-related DEGs in the developing tobacco seeds. DAF, days after flowering. Morphological appearance (b), dry weight (c), and FA content (d) of the mature seeds from the WT, NtMYB12a-OE ,NtMYB12a-RNAi , and ntmyb12a mutant plants. Asterisks represent statistically significant differences between WT and transgenic plants determined by two-tailed paired Student’s t-test (**, P < 0.01; *, P < 0.05).
FIGURE 7 NtMYB12a activates the transcription of NtLOX5 ,NtLOX6 , NtSFAR4 , and NtGDSL2 genes by binding to their promoter regions. (a) Relative expression levels of NtLOX5 ,NtLOX6 , NtSFAR4 , and NtGDSL2 genes in the35S:NtMYB12a-GR seedlings under DEX, CYC, and DEX+CYC treatment. (b) Western blotting showing the NtMYB12a-GFP band. E, eluate; I, Input. (c) The promoter structures of NtSFAR4 , NtLOX5 ,NtLOX6 , and NtGDSL2 genes. The black line represents the 3 kb sequence upstream the ATG of each target gene. The black triangles indicate the position of putative MYB recognition element (MRE) required for activation by MYB12. The while boxes represent the DNA fragments amplified in the ChIP assay. (d) ChIP enrichment test of the promoter regions bound by NtMYB12a-GFP. Asterisks represent statistically significant differences between WT and transgenic plants determined by two-tailed paired Student’s t-test (**, P < 0.01; *, P < 0.05).
FIGURE 8 NtLOX6 and NtLOX5 prevent the accumulation of FA in tobacco seeds. (a) Phylogenetic analysis of the two NtLOXs with the 6 Arabidopsis AtLOX proteins. MEGA5.0 was used to generate the neighbor-joining tree (Jones-Taylor-Thornton model). The statistical reliability of the tree topology was assessed by performing a bootstrap analysis with 1000 replicates. (b) Relative expression levels ofNtLOX5 gene in the NtLOX5-OE and NtLOX5-RNAi lines. (c) Relative expression levels of NtLOX6 gene in theNtLOX6-OE and NtLOX6-RNAi lines. (d) The FA content in the mature seeds of WT and NtLOX5 transgenic plants. (e) Percentage of PUFAs in the leaves of WT and NtLOX5 transgenic plants. (f) The FA content in the mature seeds of WT and NtLOX6 transgenic plants. (g) Percentage of PUFAs in the leaves of WT and NtLOX6transgenic plants. Values are means of three independent replicates. Error bars denote standard deviations. Asterisks represent statistically significant differences between WT and transgenic plants determined by two-tailed paired Student’s t-test (**, P < 0.01; *, P < 0.05).
FIGURE 9 Expression pattern of NtMYB12a under sucrose treatment. (a) Gus staining of theProNtMYB12a:GUS seedlings treated with 0 mM, 50 mM, 88 mM (CK), 150 mM, and 200 mM sucrose. Bar=5mm. (b) Relative expression level of NtMYB12a gene under different concentration of sucrose. Values are means of three independent replicates. Error bars denote standard deviations. The significant differences among different lines are detected by Tucky’s test. (c) Venn diagram of the DEGs from two RNA-seq anslyses. URG-OE, up-regulated genes in theNtMYB12a-OE plants; DRG-OE, down-regulated genes in theNtMYB12a-OE plants; URG-Suc, up-regulated genes by 150 mM sucrose; DRG-Suc, down-regulated genes by 150 mM sucrose.
FIGURE 10 Effect of sucrose on FA metabolism in tobacco seedlings. (a) Phenotype of WT, NtMYB12a-OE ,NtMYB12a-RNAi , and ntmyb12a mutant seedlings under sucrose treatment. Bar=1cm. (b) Relatives expression levels of NtLOX5 ,NtLOX6 , NtSFAR4 , and NtGDSL2 genes. Asterisks represent statistically significant differences between WT and transgenic plants determined by two-tailed paired Student’s t-test (**, P < 0.01; *, P < 0.05). (c) FA content of the WT,NtMYB12a-OE , NtMYB12a-RNAi , and ntmyb12a mutant seedlings. Values are means of three independent replicates. Error bars denote standard deviations. The significant differences among different lines are detected by Tucky’s test.
FIGURE 11 Schematic illustrating the regulatory network for NtMYB12a regarding promotion of flavonoids synthesis and FA degradation in tobacco.