Effects of PtoMYB61 on Lignin Biosynthesis and Salt Tolerance in Populus tomentosa

doi:10.13560/j.cnki.biotech.bull.1985.2025-0006

Abstract

Abstract:

Objective The effects of PtoMYB61 on secondary cell wall formation and stress defense of Populus tomentosa were studied, so as to lay a foundation for studying wood development and stress resistance breeding. Method RT-PCR was used to clone the PtoMYB61 gene from P. tomentosa. Phylogenetic tree analysis and homology comparison was to predict its biological function. RT-qPCR was applied to analyze the tissue expression specificity of PtoMYB61 and its response to biotic and abiotic stresses. Leaf disc method was to transform P. tomentosa and the lines of overexpressed and knocked-out PtoMYB61 were obtained transformed. Tissue section, toluidine blue staining, lignin content determination, and expressions of key enzyme genes in secondary cell wall biosynthesis pathway were used to analyze the effect of PtoMYB61 on poplar secondary development. Transgenic lines were treated with 150 mmol/L NaCl, and the effects of PtoMYB61 on the tolerance of P. tomentosa to salt were analyzed by phenotypic observation and physiological index determination. Result PtoMYB61 gene encodes a R2R3-MYB transcription factor consisting of 309 amino acids, which is highly expressed in axillary buds, leaves and stems, and is induced by salt, ABA and fungal diseases. Overexpressed and knocked-out lines have no significant difference in growth phenotype compared with wild type, but the number of xylem cells in the overexpressed plants of PtoMYB61 increase, the lignin content significantly increases, and the expressions of key enzymes in secondary cell wall biosynthesis are up-regulated, while knocking PtoMYB61 out leads to the decrease of xylem cell layers, the decrease of lignin content and the down-regulation of key enzymes in secondary cell wall biosynthesis. Salt treatment shows that compared with wild type, PtoMYB61-knockout plants are more sensitive to salt stress, while the overexpressed plants have certain tolerance. Conclusion PtoMYB61 responds to the induction of salt stress and influences the tolerance of poplar to salt stress by regulating the development of secondary cell wall.

Key words: poplar, transcription factor, PtoMYB61, secondary cell wall, salt tolerance

LI Xiao-huan, CHEN Xiang-yu, TAO Qi-yu, ZHU Ling, TANG Ming, YAO Yin-an, WANG Li-jun. Effects of PtoMYB61 on Lignin Biosynthesis and Salt Tolerance in Populus tomentosa[J]. Biotechnology Bulletin, 2025, 41(6): 284-296.

Figures/Tables 8

Fig. 1 Phylogenetic analysis and amino acid sequence alignment of PtoMYB61A: Protein phylogenetic tree analysis of PtoMYB61; B: amino acid homology analysis of PtoMYB61

Fig. 2 Specificity analysis of PtoMYB61 expression patternA: Quantitative analysis of the expression of PtoMYB61 in different tissues by fluorescence, R: root, S: stem, YL: young leaf, ML: mature leaf, P: petiole, AB: terminal bud, LB: axillary bud. Asterisks indicate significant differences using Student’s t-test ( *P<0.05; **P<0.01 ), n=3, the same below

Fig. 3 Positive identification of transgenic plants of PtoMYB61A: Positive identification electropherogram of 35S::PtoMYB61 overexpressed plants, different numbers represent independent transgenic lines; B: RT-qPCR quantitative results of overexpressed strains; C: schematic diagram of CRISPR/Cas9 targeted editing of PtoMYB61, T1 to T3 represent the design areas of three targets; D: Sequencing analysis of PtoMYB61 mutant lines, the picture shows the base deletion in the middle of 3, 4 and 5 independent lines

Fig. 4 Morphological observation of transgenic lines of PtoMYB61A: 9-week-old Populus tomentosa, scale bar=10 cm; B: roots of 9-week-old P. tomentosa plant, scale bar=5 cm; C: height statistics of P. tomentosa from 4 weeks to 9 weeks; D: statistics of stem diameter of P. tomentosa 5 cm above the ground from 4 weeks to 9 weeks; E: the root length of P. tomentosa growing for 11 weeks; F: root surface area of P. tomentosa growing for 11 weeks

Fig. 5 Detection of lignin and total flavonoids in transgenic lines of PtoMYB61A: Cross-section of the stem of the 5th and 7th internode of WT plant and PtoMYB61 transgenic line under the stereomicroscope; B: statistics of xylem cell layers in the 4th, 5th and 7th stem nodes, n=10; C: lignin content in stems of WT and transgenic plants, n=3; D: total flavonoids content in leaves of WT and transgenic plants. The error line indicates the mean SD, and different lowercase letters indicate the significant difference of Tukey’s post hoc test based on one-way ANOVA (P<0.05), the same below

Fig. 6 Expression analysis of secondary cell wall biosynthesis genes in transgenic poplar stemsA: Lignin biosynthesis genes; B: cellulose biosynthesis genes; C: xylan synthase gene

Fig. 7 Phenotype of PtoMYB61 over-expressing and knocking-out lines under salt stressA: Phenotypic map after 7 d of 150 mmol/L NaCl treatment, bar=10 cm; B: DAB staining results after four days of 150 mmol/L NaCl treatment, bar=5 cm

Fig. 8 Physiological indexes of PtoMYB61 before and after salt stress

References 37

1	Sundell D, Street NR, Kumar M, et al. AspWood: high-spatial-resolution transcriptome profiles reveal uncharacterized modularity of wood formation in Populus tremula [J]. Plant Cell, 2017, 29(7): 1585-1604.
2	Cosgrove DJ, Jarvis MC. Comparative structure and biomechanics of plant primary and secondary cell walls [J]. Front Plant Sci, 2012, 3: 204.
3	Kelly SM, Munoz-Munoz J, van Sinderen D. Plant glycan metabolism by bifidobacteria [J]. Front Microbiol, 2021, 12: 609418.
4	Weng JK, Chapple C. The origin and evolution of lignin biosynthesis [J]. New Phytol, 2010, 187(2): 273-285.
5	Zhao Q. Lignification: flexibility, biosynthesis and regulation [J]. Trends Plant Sci, 2016, 21(8): 713-721.
6	Ohtani M, Demura T. The quest for transcriptional hubs of lignin biosynthesis: beyond the NAC-MYB-gene regulatory network model [J]. Curr Opin Biotechnol, 2019, 56: 82-87.
7	Ko JH, Kim WC, Han KH. Ectopic expression of MYB46 identifies transcriptional regulatory genes involved in secondary wall biosynthesis in Arabidopsis [J]. Plant J, 2009, 60(4): 649-665.
8	McCarthy RL, Zhong RQ, Ye ZH. MYB83 is a direct target of SND1 and acts redundantly with MYB46 in the regulation of secondary cell wall biosynthesis in Arabidopsis [J]. Plant Cell Physiol, 2009, 50(11): 1950-1964.
9	Geng P, Zhang S, Liu JY, et al. MYB20, MYB42, MYB43, and MYB85 regulate phenylalanine and lignin biosynthesis during secondary cell wall formation [J]. Plant Physiol, 2020, 182(3): 1272-1283.
10	Zhong RQ, Ye ZH. MYB46 and MYB83 bind to the SMRE sites and directly activate a suite of transcription factors and secondary wall biosynthetic genes [J]. Plant Cell Physiol, 2012, 53(2): 368-380.
11	张勇, 张守攻, 齐力旺, 等. 杨树——林木基因组学研究的模式物种 [J]. 植物学通报, 2006, 41(3): 286-293.
	Zhang Y, Zhang SG, Qi LW, et al. Poplar as a model for forest tree in genome research [J]. Chin Bull Bot, 2006, 41(3): 286-293.
12	Zhong RQ, McCarthy RL, Haghighat M, et al. The poplar MYB master switches bind to the SMRE site and activate the secondary wall biosynthetic program during wood formation [J]. PLoS One, 2013, 8(7): e69219.
13	Chai GH, Qi G, Cao YP, et al. Poplar PdC3H17 and PdC3H18 are direct targets of PdMYB3 and PdMYB21, and positively regulate secondary wall formation in Arabidopsis and poplar [J]. New Phytol, 2014, 203(2): 520-534.
14	McCarthy RL, Zhong RQ, Fowler S, et al. The poplar MYB transcription factors, PtrMYB3 and PtrMYB20, are involved in the regulation of secondary wall biosynthesis [J]. Plant Cell Physiol, 2010, 51(6): 1084-1090.
15	Zhong RQ, McCarthy RL, Lee CH, et al. Dissection of the transcriptional program regulating secondary wall biosynthesis during wood formation in poplar [J]. Plant Physiol, 2011, 157(3): 1452-1468.
16	Wang LJ, Lu WX, Ran LY, et al. R2R3-MYB transcription factor MYB6 promotes anthocyanin and proanthocyanidin biosynthesis but inhibits secondary cell wall formation in Populus tomentosa [J]. Plant J, 2019, 99(4): 733-751.
17	Yang L, Zhao X, Ran LY, et al. PtoMYB156 is involved in negative regulation of phenylpropanoid metabolism and secondary cell wall biosynthesis during wood formation in poplar [J]. Sci Rep, 2017, 7: 41209.
18	Wang ZF, Mao YL, Guo YJ, et al. MYB transcription Factor161 mediates feedback regulation of Secondary wall-associated NAC-Domain1 family genes for wood formation [J]. Plant Physiol, 2020, 184(3): 1389-1406.
19	Jiao B, Zhao X, Lu WX, et al. The R2R3 MYB transcription factor MYB189 negatively regulates secondary cell wall biosynthesis in Populus [J]. Tree Physiol, 2019, 39(7): 1187-1200.
20	Tang XF, Zhuang YM, Qi G, et al. Poplar PdMYB221 is involved in the direct and indirect regulation of secondary wall biosynthesis during wood formation [J]. Sci Rep, 2015, 5: 12240.
21	Guo HY, Wang YC, Wang LQ, et al. Expression of the MYB transcription factor gene BplMYB46 affects abiotic stress tolerance and secondary cell wall deposition in Betula Platyphylla [J]. Plant Biotechnol J, 2017, 15(1): 107-121.
22	Yu Y, Liu HZ, Zhang N, et al. The BpMYB4 transcription factor from Betula platyphylla contributes toward abiotic stress resistance and secondary cell wall biosynthesis [J]. Front Plant Sci, 2021, 11: 606062.
23	Hu RB, Qi G, Kong YZ, et al. Comprehensive analysis of NAC domain transcription factor gene family in Populus trichocarpa [J]. BMC Plant Biol, 2010, 10: 145.
24	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔct Method [J]. Methods, 2001, 25(4): 402-408.
25	Shi R, Sun YH, Li QZ, et al. Towards a systems approach for lignin biosynthesis in Populus trichocarpa: transcript abundance and specificity of the monolignol biosynthetic genes [J]. Plant Cell Physiol, 2010, 51(1): 144-163.
26	Zhao YQ, Song XQ, Zhou HJ, et al. KNAT2/6b, a class I KNOX gene, impedes xylem differentiation by regulating NAC domain transcription factors in poplar [J]. New Phytol, 2020, 225(4): 1531-1544.
27	Ma XL, Zhang QY, Zhu QL, et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants [J]. Mol Plant, 2015, 8(8): 1274-1284.
28	汪丽君. 杨树转录因子MYB6调控类黄酮和木质素生物合成的机制研究 [D]. 重庆: 西南大学, 2018.
	Wang LJ. The transcriptional regulatory mechanism of the poplar transcription factor MYB6 involved in flavonoid and lignin biosynthesis [D]. Chongqing: Southwest University, 2018.
29	任园宇, 魏东伟, 王中伟, 等. 亚硝酸钠-硝酸铝比色法测定干旱胁迫前后玉米幼苗的总黄酮含量 [J]. 农学学报, 2020, 10(5): 15-20.
	Ren YY, Wei DW, Wang ZW, et al. Total flavonoids in maize seedlings before and after drought stress: determination with sodium nitrite-aluminum nitrate colorimetry [J]. J Agric, 2020, 10(5): 15-20.
30	Rao XL, Dixon RA. Current models for transcriptional regulation of secondary cell wall biosynthesis in grasses [J]. Front Plant Sci, 2018, 9: 399.
31	Zhong RQ, Cui DT, Ye ZH. Secondary cell wall biosynthesis [J]. New Phytol, 2019, 221(4): 1703-1723.
32	Novaković L, Guo TT, Bacic A, et al. Hitting the wall-sensing and signaling pathways involved in plant cell wall remodeling in response to abiotic stress [J]. Plants, 2018, 7(4): 89.
33	Zhang J, Wang XQ, Wang HT, et al. Overexpression of reduced wall acetylation c increases xylan acetylation and biomass recalcitrance in Populus [J]. Plant Physiol, 2023, 194(1): 243-257.
34	Zhang J, Tuskan GA, Tschaplinski TJ, et al. Transcriptional and post-transcriptional regulation of lignin biosynthesis pathway genes in Populus [J]. Front Plant Sci, 2020, 11: 652.
35	Wang LJ, Ran LY, Hou YS, et al. The transcription factor MYB115 contributes to the regulation of proanthocyanidin biosynthesis and enhances fungal resistance in poplar [J]. New Phytol, 2017, 215(1): 351-367.
36	Vanholme R, De Meester B, Ralph J, et al. Lignin biosynthesis and its integration into metabolism [J]. Curr Opin Biotechnol, 2019, 56: 230-239.
37	齐琪, 马书荣, 徐维东. 盐胁迫对植物生长的影响及耐盐生理机制研究进展 [J]. 分子植物育种, 2020, 18(8): 2741-2746.
	Qi Q, Ma SR, Xu WD. Advances in the effects of salt stress on plant growth and physiological mechanisms of salt tolerance [J]. Mol Plant Breed, 2020, 18(8): 2741-2746.

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