Abstract
Key message
MANNANASE7 gene in Brassica napus L. encodes a hemicellulose which located at cell wall or extracellular space and dehiscence-resistance can be manipulated by altering the expression of MANNANASE7.
Abstract
Silique dehiscence is an important physiological process in plant reproductive development, but causes heavy yield loss in crops. The lack of dehiscence-resistant germplasm limits the application of mechanized harvesting and greatly restricts the rapeseed (Brassica napus L.) production. Hemicellulases, together with cellulases and pectinases, play important roles in fruit development and maturation. The hemicellulase gene MANNANASE7 (MAN7) was previously shown to be involved in the development and dehiscence of Arabidopsis (Arabidopsis thaliana) siliques. Here, we cloned BnaA07g12590D (BnMAN7A07), an AtMAN7 homolog from rapeseed, and demonstrate its function in the dehiscence of rapeseed siliques. We found that BnMAN7A07 was expressed in both vegetative and reproductive organs and significantly highly expressed in leaves, flowers and siliques where the abscission or dehiscence process occurs. Subcellular localization experiment showed that BnMAN7A07 was localized in the cell wall. The biological activity of the BnMAN7A07 protein isolated and purified through prokaryotic expression system was verified to catalyse the decomposition of xylan into xylose. Phenotypic studies of RNA interference (RNAi) lines revealed that down-regulation of BnMAN7A07 in rapeseed could significantly enhance silique dehiscence-resistance. In addition, the expression of upstream silique development regulators is altered in BnMAN7A07-RNAi plants, suggesting that a possible feedback regulation mechanism exists in the regulation network of silique dehiscence. Our results demonstrate that dehiscence-resistance can be manipulated by altering the expression of hemicellulase gene BnMAN7A07, which could provide an available genetic resource for breeding practice in rapeseed which is beneficial to mechanized harvest.
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This work was supported by the National Key R&D Program of China (2016YFD0101900 and 2016YFD0100305).
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XLT, YLL and YKY designed the experiments. YKY, YLL, KMZ, LND, ZW, YHY and JC performed the experiments and analysed the data. YLL and YKY wrote the paper. YLL, YKY, LZX, YML and XLT revised the paper. All authors read and approved the final manuscript.
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Fig. S1 The installation of quantitative determination of the fore for silique dehiscence The siliques were glued to a plate, and the replum was paralleled to the plate. One side of L-shaped hook was fixed in the probe of the texture analyser (TAXT2-HD, Stable Micro System, UK), another side hooked the pedicel at the joint point of silique and pedicel. During the test, the plate was held in hands, the probe was moved upwards at the speed of 2 mm/min and the silique was opened. At the same time, the probe recorded the opening strength (TIF 34311 KB)
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Fig. S2 Expression pattern of MAN7 homologous genes in B. napus. A total of 7 homologs were obtained from the B. napus genome based on the BLAT search in B. napus genomic database using AtMAN7 protein (NP_201447.1) sequence. The expression level of these genes in different tissues is compared reference to Fragments Per Kilobase per Million (FPKM). The numbers in the table represent the FPKM and the highlighted BnaA07g12590D, which showed the highest expression level among 7 homologs was selected as the target gene in present study. This table is organized according to the BrassicaEDB (https://biodb.swu.edu.cn/brassica/). (TIF 5565 KB)
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Fig. S3 Phylogenetic relationship of BnMAN7A07 with other closely related MAN7 proteins. A phylogenetic tree of 6 MAN7 proteins from Brassica napus, Brassica rapa, Raphanus sativus, Sisymbrium officinale and Arabidopsis thaliana was generated in MEGA7 software using the neighbor-joining (NJ) method. A total of 500 boots trap replicates were performed and values are indicated above the branch points (TIF 5646 KB)
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Fig. S4 Bioinformatic analyses of BnMAN7A07 protein. a Signal peptide prediction of BnMAN7A07. SignalP-5.0 Server was used to predict the signal peptide of BnMAN7A07 protein. It showed that the cleavage site is between 25 and 26 amino acids with 0.9587 reliability. SP: Signal peptide score; CS: the cleavage site and OTHER: the probability that the sequence does not have any kind of signal peptide. b Subcellular localization prediction of BnMAN7A07. The subcellular localization of BnMAN7A07 protein was predicted by Euk-mPLoc2.0 Server. It predicted BnMAN7A07 localized at extra cell (TIF 12003 KB)
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Fig. S5 The standard curve for the xylose content calculation. Different amount of xylose (0, 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg, 1 mg) was mix with NaAc buffer and DNS. The solution was boiled at 100 ℃ in water bath for 5 min. Add total volume to 25ml with ddH2O and OD540 absorption value was detected by spectrophotometer. The standard curve was performed using Excel (TIF 4639 KB)
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Fig. S6 PCR analysis of positive transformed plants using NPT-II-specific primers. The number represent different RNAi lines. +: the plasmid as template, -: ddH2O as template (TIF 4064 KB)
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Fig. S7 Time-force curve record exterior forces necessary for silique dehiscence. The pulling force on the silique increased continuously along with the probe moving, and when the silique ripped, the force on the silique rapidly decreased, and the peak of the curve record the max exterior force that was needed for the silique dehiscence (TIF 28881 KB)
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Fig. S8 Significance analysis of the force for opening the siliques from different lines. The number represent different siliques form each plant. The significances of the force differences between WT, BnMAN7A07-RNAi line 2 and 3 and are indicated (Student’s t-test, *0.01 < P < 0.05, **** P < 0.0001) (TIF 2387 KB)
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Li, YL., Yu, YK., Zhu, KM. et al. Down-regulation of MANNANASE7 gene in Brassica napus L. enhances silique dehiscence-resistance. Plant Cell Rep 40, 361–374 (2021). https://doi.org/10.1007/s00299-020-02638-5
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DOI: https://doi.org/10.1007/s00299-020-02638-5