Fig 1.
Amino acid sequence alignment of BnLATE and Arabidopsis LATE.
The C2H2 zinc-finger domain, LDLXL domain, and B-box indicated by solid black, dotted black, and dotted red boxes, respectively. Numbers in parentheses indicate the amino acid position in each protein.
Fig 2.
Molecular phylogenetic tree and schematic functional domains of the Cys2/His2-type zinc-finger proteins.
(A) Molecular phylogeny of the Cys2/His2 type zinc-finger proteins. BnLATE and AtLATE clustered into a group. Unrooted neighbor-joining tree of the C-terminal end of amino acid sequences encompassing the predicted mature domain. Branch lengths scaled to the number of amino acid changes indicated on the scale bar. (B) Schematic functional domains of the Cys2/His2 type zinc-finger proteins. I and II denote two clades. Conserved motifs identified within amino acid sequences using the online MEME (http://meme.nbcr.net/meme/cgi-bin/meme.cgi). The motifs and their corresponding positons were represented by different colors of square. The parameters of MEME were set as following: optimum width 10–100 amino acids, the max number of any repeated motifs was set at 25. (C) Conserved motif models identified with MEME procedure among members of LATE family from B. napus and Arabidopsis. Motifs 1, 2, and 4 are zinc finger domain, motifs 3, 5, and 6 are L-box, EAR-like domain, and B-box respectively.
Fig 3.
Phenotypic variations caused by BnLATE overexpression in Arabidopsis.
(A) GUS activity was detectable in silique of pBnLATE::GUS transgenic Arabidopsis. The dotted black line boxes showed that amplification of silique in the red line boxes. Bar, 0.50 cm. (B) Expression level of BnLATE in the silique of D35S-BnLATE transgenic lines was revealed by Real-time PCR. Expression level of BnLATE and AtLATE was determined relative to that of the internal control β-AtACTIN2 (AT3G18780), multiplied by 10. The experiment was performed in triplicate. Significance was determined with Student’s t-test. Asterisks above the columns indicate the significant difference compared to Col-0 and EV. * p< 0.05, ** P< 0.01. (C) Representative images of yellowed siliques of controls Col-0, EV and D35S-BnLATE transgenic lines (T5). Bar, 0.50 cm
Fig 4.
Histological analysis of 15-DAP silique cross-sections of D35S::BnLATE transgenic Arabidopsis lines.
DAP: days after pollination. (A) Microscopy observations of cross-sections of D35S::BnLATE transgenic Arabidopsis siliques stained with Safranin/fast green. A representative result of five independent experiments is shown. Bars, 100 μm. (B) Microscopy observations of the stained replum. Bar, 50 μm. (C) Microscopy observations of the stained carpel. Bar, 10 μm. (D) Determination lignin content in the 15-DAP siliques of D35S::BnLATE transgenic Arabidopsis lines. The experiment was performed in triplicate, and results represent the mean ± S.D. Significance was determined with Student’s t-test. Asterisks above the columns indicate the significant difference compared to Col-0 and EV. * p< 0.05, ** p< 0.01. Abbreviations: en, endocarp; ex, exocarp; m, mesocarp; r, replum; se, septum; sl, separation layer; v, vascular bundle; vm, valve margin.
Table 1.
Percentage of D35S::BnLATE transgenic Arabidopsis, Col-0 and EV plants exhibiting silique shattering resistance.
Fig 5.
Histological analysis and expression level of BnLATE in silique of B. napus accessions 73290.
(A) BnLATE expression level was examined by RT-PCR. β-BnACTIN2 was used for standardization of the template concentration. (B) Microscopic observations of the replum and carpel stained by safranin/fast green. One representation of five independent experiments is shown. 12, 24, 36, and 48 DAP from left to right. Bars, 100 μm. (C) Determination of lignin content in the silique wall of 73290 at 12, 24, 36, 48, and 60 DAP. (D) Silique shattering-resistance index (SRI) of the mature silique of 73290 was determinated by a random impact test. The test was performed in triplicate. Significance was defined with Student’s t-test. I, II, III, IV and V showed that five SRI from high to low. Abbreviations: en, endocarp; ena, endocarp a; enb, endocarp b; m, mesocarp; r, replum; se, septum; sl, separation layer; v, vascular bundle; vm, valve margin.
Fig 6.
Differentially expressed genes in overexpressing BnLATE Arabidopsis lines.
(A) Functional categories of differentially expressed genes (DEGs) in a D35S::BnLATE transgenic Arabidopsis line. (B) Mapped DEGs on KEGG biological pathways. (C) Down-regulated DEGs encoding enzymes in a D35S::BnLATE transgenic Arabidopsis line L8. Red dashed box indicates the preferred pathways. (D) Up-regulated DEGs encoding enzymes in a D35S::BnLATE transgenic Arabidopsis line L8. Red dashed box indicates the preferred pathways.
Fig 7.
Schematic diagram of lignification and the expression pattern of genes involved in lignin biosynthesis in wild-type Col-0 and D35S::BnLATE transgenic Arabidopsis lines.
(A) Validation by real-time PCR of representative DEGs that are down-regulated in the siliques of D35S::BnLATE transgenic Arabidopsis lines (L21 and L8) and Col-0. Results represent the mean ± S.D from three replicates. (B) Validation by real-time PCR of representative DEGs that are up-regulated in the siliques of D35S::BnLATE transgenic Arabidopsis lines (L8 shown) and Col-0. The expression level was determined relative to that of the internal control, β-AtACTIN2 (AT3g18780). The experiment was performed in triplicate, and results represent the mean ± S.D. Significance was determined using Student’s t-test. Asterisks indicate significant differences between Col-0 and EV. * p< 0.05, ** p< 0.01. (C) A schematic model of lignin and flavonoid biosynthesis in Arabidopsis.
Table 2.
Differentially expressed genes involved in the lignin polymerization.
Fig 8.
BnLATE expression variation and silique morphology of pBnLATE::BnLATE transgenic Arabidopsis lines.
(A) Validation of target insert using specific PCR primers in 11 independent pBnLATE::BnLATE Arabidopsis lines (1–11). (B) Silique morphology at four developing stages 5 DAP (I), 10 DAP (II), 20 DAP (III), and 30 DAP (IV). Bar, 0.50 cm. (C) Expression pattern of BnLATE in silique of pBnLATE::BnLATE transgenic lines using Real-time PCR. Expression level of BnLATE was determined relative to that of the internal control β-AtACTIN2 (AT3g18780), multiplied by 10. The experiment was performed in triplicate. (D) Representative yellowed siliques of Col-0 and pBnLATE::BnLATE transgenic lines. Bar, 0.50 cm. (E) Average percentage of high-, middle- and low-shattering-resistance siliques of pBnLATE::BnLATE transgenic lines, three biological replicates with 30 siliques each. Silique of Col-0 shows shattering-resistance. Asterisks above the columns indicate the significant difference vs. Col-0. * p< 0.05, ** p< 0.01. (F) Expression pattern of four representative peroxidase genes (PRX2, PRX35, PRX71, and PRX72) in siliques of pBnLATE::BnLATE-1 (T3) and Col-0 by real-time PCR. Expression level was determined relative to that of the internal control β-AtACTIN2 (AT3G18780). The experiment was performed in triplicate. Significance was defined with Student’s t-test. Asterisks above the columns indicate the significant difference compared with Col-0 and EV. * p< 0.05, ** P< 0.01.
Fig 9.
Histological observation on silique of pBnLATE::BnLATE transgenic lines.
One representative result of five independent experiments is shown. Cross-sections of silique replum and carpel of pBnLATE::BnLATE and Col-0 at stages I, II, III and IV were performed and stained with safranin/fast green. In contrast to Col-0, no obvious staining color (red) was observed in the carpel of pBnLATE::BnLATE at stages III and IV, and lignification cells in the dehiscence zone was apparently less as well. Abbreviations: en, endocarp; ex, exocarp; m, mesocarp; r, replum; se, septum; v, vascular bundle; vm, valve margin. Bars, 50 μm.