LBD12转录因子调控毛果杨木材形成研究

高源; 孙佳彤; 周晨光; 姜立泉; 李伟; 李爽

doi:10.12302/j.issn.1000-2006.202301002

高源, 孙佳彤, 周晨光, 姜立泉, 李伟, 李爽

南京林业大学学报（自然科学版） ›› 2024, Vol. 48 ›› Issue (1) : 29-38.

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南京林业大学学报（自然科学版） ›› 2024, Vol. 48 ›› Issue (1) : 29-38. DOI: 10.12302/j.issn.1000-2006.202301002

专题报道Ⅰ:基因编辑与分子设计育种专题(执行主编施季森尹佟明)

LBD12转录因子调控毛果杨木材形成研究

高源 ¹ ,
孙佳彤 ² ,
周晨光 ¹ ,
姜立泉 ¹ ,
李伟 ¹ ,
李爽 ¹^,^*

作者信息 +

Regulation of LBD12 transcription factor on wood formation in Populus trichocarpa

GAO Yuan ¹ ,
SUN Jiatong ² ,
ZHOU Chenguang ¹ ,
CHIANG Vincent ¹ ,
LI Wei ¹ ,
LI Shuang ¹^,^*

Author information +

文章历史 +

摘要

【目的】创制毛果杨(Populus trichocarpa)LBD12(lateral organ boundaries domain 12)转录因子的过量表达植株,分析表型特征,初步探究PtrLBD12转录因子在毛果杨木材形成中的生物功能。【方法】基于前期的研究,筛选出了木材形成关键调控因子PtrbHLH186调控的下游转录因子PtrLBD12,通过基因克隆获得PtrLBD12基因;创制PtrLBD12过量表达毛果杨植株,测定并统计生长30、60、90 d毛果杨野生型和过表达植株的生长指标;对生长120 d的转基因及野生型植株各茎节进行石蜡切片、化学染色观察及不同细胞类型的统计分析;通过定量PCR测定过表达PtrLBD12基因对22个木质素单体合成酶基因表达水平的影响。【结果】获得毛果杨PtrLBD12过表达植株;与野生型相比,转基因植株的株高、地径等生长指标水平明显降低;纤维和导管细胞的数量显著增多,导管细胞的孔径减小,形成层细胞的形态结构无明显变化;木质素沉积水平增加,木质化程度增强;PtrLBD12的过量表达促进了木质素合成酶基因的表达。【结论】LBD12转录因子在毛果杨木材形成过程中发挥重要的调控作用,可调节木质素单体合成酶基因的表达,改变木质素沉积方式。此外,该基因也可以改变木质部细胞形态结构,影响植株生长发育。

Abstract

【Objective】The formation of wood, one of the most important raw materials for pulp and energy, depends on a complex and precise transcriptional regulation process in which transcription factors figure prominently. In Populus trichocarpa, PtrLBD12 (lateral organ boundaries domain 12) is a transcription factor lying downstream of PtrbHLH186, a key regulator of wood formation. Hence, PtrLBD12 was studied in depth here to investigate its function in tree growth and wood formation.【Method】 To determine the role of the PtrLBD12 transcription factor in the growth and wood formation of poplar trees, we analyzed its expression characteristics, generated PtrLBD12 overexpressing plants of P. trichocarpa, and assessed the growth and wood formation traits of transgenic plants. (1) Xylem, phloem, and terminal bud and leaf samples of wild-type P. trichocarpa plants, cultivated in a greenhouse, were collected to extract their respective RNA. Next, transcriptome sequencing was done to analyze the patterns of differentially expressed genes in those distinct tissues, as well as their expression levels of PtrLBD12. (2) To clarify the expression pattern of the PtrLBD12 protein, its subcellular localization was investigated by using the transient transformation system of stem-differentiating xylem protoplasts of P. trichocarpa. (3) To create PtrLBD12-overexpressing plants, the Agrobacterium tumefaciens-mediated transformation system of P. trichocarpa was used, with transgenic plants identified at both the DNA and RNA level. (4) Plant stem height, ground (basal) stem diameter, number of stem nodes, and length of the 8th stem node of transgenic and wild-type plants were measured at 30, 60 and 90 days after planting. (5) The 2nd, 4th, 6th and 8th stem segments of overexpressing and wild-type greenhouse plants cultivated for 4 months were paraffin-sectioned. Their stem characteristics were then respectively observed via Safranin O/Fast Green and Toluidine Blue staining. LAS X V2.0 software was used to calculate the number of vessel and fiber cells, as well as average lumen area of vessel. (6) The relative expression levels of 22 monolignol biosynthetic pathway genes in the PtrLBD12-overexpressing plants were determined by using quantitative PCR and applying the 2^△△Ct calculation.【Result】 (1) Transcriptome analysis of the xylem, phloem, and terminal bud and leaf tissues of P. trichocarpa revealed a higher expression level of PtrLBD12 in both the xylem and phloem. (2) Subcellular localization showed that PtrLBD12 was expressed in the nucleus, where transcription factors in general were found. (3) Three PtrLBD12 overexpressing transgenic lines of P. trichocarpa were obtained, whose relative expression levels were 40.91, 79.51 and 102.19. (4) Overexpressing PtrLBD12 adversely affected the normal growth and development of P. trichocarpa: plant height, ground stem diameter, number of stem nodes, and length of the 8th stem node all decreased significantly in transgenic plants vis-à-vis the wild type. (5) However, overexpression of PtrLBD12 did lead to significantly more vessel and fiber cells per unit of area in the stems of transgenic plants, but these vessels had smaller lumen area. Furthermore, the augmented expression of PtrLBD12 enhanced lignification of the plant stem. (6) Finally, PtrLBD12’s overexpression bolstered the expression levels of multiple genes in the monolignol biosynthetic pathway, namely PtrPAL1, PtrC4H1, PtrC4H2, PtrHCT6, PtrCSE, PtrCSE2, PtrCCoAOMT1, PtrCCoAOMT2, PtrCCoAOMT3, PtrCCR2 and PtrCAld5H1.【Conclusion】As the gene downstream of PtrbHLH186, a key regulator of wood formation in poplar, the LBD12 transcription factor is able to govern the expression of monolignol biosynthetic genes, change the mode of lignin deposition, alter the morphology of xylem cells, and thereby affect plant growth and development.

导出引用

高源, 孙佳彤, 周晨光, 等. LBD12转录因子调控毛果杨木材形成研究[J]. 南京林业大学学报（自然科学版）. 2024, 48(1): 29-38 https://doi.org/10.12302/j.issn.1000-2006.202301002

GAO Yuan, SUN Jiatong, ZHOU Chenguang, et al. Regulation of LBD12 transcription factor on wood formation in Populus trichocarpa[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2024, 48(1): 29-38 https://doi.org/10.12302/j.issn.1000-2006.202301002

中图分类号： S792.11;Q943.2

参考文献

列表( 原文顺序 | 文献年度倒序 | 文中引用次数倒序 ) 可视化分析

[1]	ZHANG J, NIEMINEN K, SERRA J A, et al. The formation of wood and its control[J]. Current Opinion Plant Biology, 2014, 17: 56-63. DOI: 10.1016/j.pbi.2013.11.003. 本文引用 [1]

[2]	SIEDLECKA A, WIKLUND S, PÉRONNE MA, et al. Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of Populus[J]. Plant Physiology, 2008, 146(2): 554-565. DOI: 10.1104/pp.107.111963. 本文引用 [1]

[3]

孙滨, 占小登, 曹立勇, 等. 水稻AP2/ERF转录因子的研究进展[J]. 农业生物技术学报, 2017, 25(11): 1860-1869.

SUN

, ZHAN

X D

, CAO

L Y

, et al. Research progress of AP2/ERF transcription factor in rice (Oryza sativa)[J]. Journal of Agricultural Biotechnology, 2017, 25(11): 1860-1869. DOI: 10.3969/j.issn.1674-7968.2017.11.013.

本文引用 [1]

[4]	姜秀明. 番茄HD-Zip转录因子家族生物信息学分析及HD-ZipⅠ亚族抗逆相关基因鉴定分析[D]. 哈尔滨: 东北农业大学, 2017. JIANG X M. Bioinformatics analysis of HD-zip gene family and identification of HD-zip ⅠGenes related to stress resistance in tomato[D]. Harbin:Northeast Agricultural University, 2017. 本文引用 [1]

[5]	毛昱力. 毛果杨PtrDLT和PtrWOX4基因在维管形成层发育中的功能解析[D]. 哈尔滨: 东北林业大学, 2020. DOI: 10.27009/d.cnki.gdblu.2020.000763. MAO Y L. Functional analysis of PtrDLT and PtrWOX4 genes in the development of vascular cambium in Populus trichocarpa[D]. Harbin:Northeast Forestry University, 2020. 本文引用 [1]

[6]	CHEN H, WANG J P, LIU H Z, et al. Hierarchical transcription factor and chromatin binding network for wood formation in black cottonwood (Populus trichocarpa)[J]. Plant Cell, 2019, 31(3): 602-626. DOI: 10.1105/tpc.18.00620. 本文引用 [2]

[7]	MCCARTHY R L, ZHONG R Q, YE Z H. 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 Physiology, 2009, 50(11): 1950-1964. DOI: 10.1093/pcp/pcp139. 本文引用 [1]

[8]	WANG Z F, MAO Y L, GUO Y J, et al. MYB transcription Factor161 mediates feedback regulation of Secondary wall-associated NAC-Domain1 family genes for wood formation[J]. Plant Physiology, 2020, 184(3): 1389-1406. DOI: 10.1104/pp.20.01033. 本文引用 [3]

[9]	ZHONG R Q, LEE C H, YE Z H. Functional characterization of poplar wood-associated NAC domain transcription factors[J]. Plant Physiology, 2010, 152(2): 1044-1055. DOI: 10.1104/pp.109.148270. 本文引用 [1]

[10]	ZHONG R Q, MCCARTHY R L, LEE C H, et al. Dissection of the transcriptional program regulating secondary wall biosynthesis during wood formation in poplar[J]. Plant Physiology, 2011, 157(3): 1452-1468. DOI: 10.1104/pp.111.181354. 本文引用 [1]

[11]	OHTANI M, NISHIKUBO N, XU B, et al. A NAC domain protein family contributing to the regulation of wood formation in poplar[J]. Plant J, 2011, 67(3): 499-512. DOI: 10.1111/j.1365-313X.2011.04614.x. 本文引用 [1]

[12]	LI Q Z, LIN Y C, SUN Y H, et al. Splice variant of the SND1 transcription factor is a dominant negative of SND1 members and their regulation in Populus trichocarpa[J]. PNAS, 2012, 109(36): 14699-14704. DOI: 10.1073/pnas.1212977109. 本文引用 [1]

[13]	LIN Y C, LI W, SUN Y H, et al. SND1 transcription factor-directed quantitative functional hierarchical genetic regulatory network in wood formation in Populus trichocarpa[J]. Plant Cell, 2013, 25(11): 4324-4341. DOI: 10.1105/tpc.113.117697. 本文引用 [1]

[14]	LU S F, LI Q Z, WEI H R, et al. Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa[J]. PNAS, 2013, 110(26): 10848-10853. DOI: 10.1073/pnas.1308936110. 本文引用 [1]

[15]	YEH C S, WANG Z F, MIAO F, et al. A novel synthetic-genetic-array-based yeast one-hybrid system for high discovery rate and short processing time[J]. Genome Research, 2019, 29(8): 1343-1351. DOI: 10.1101/gr.245951.118. 本文引用 [2]

[16]	ZHONG R Q, LEE C H, ZHOU J L, et al. A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis[J]. Plant Cell, 2008, 20(10): 2763-2782. DOI: 10.1105/tpc.108.061325. 本文引用 [1]

[17]	ZHOU J L, LEE C H, ZHONG R Q, et al. MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis[J]. Plant Cell, 2009, 21(1): 248-266. DOI: 10.1105/tpc.108.063321. 本文引用 [1]

[18]	KUBO M, UDAGAWA M, NISHIKUBO N, et al. Transcription switches for protoxylem and metaxylem vessel formation[J]. Genes and Development, 2005, 19(16): 1855-1860. DOI: 10.1101/gad.1331305. 本文引用 [1]

[19]	ZHONG R Q, RICHARDSON E A, YE Z H. Two NAC domain transcription factors, SND1 and NST1, function redundantly in regulation of secondary wall synthesis in fibers of Arabidopsis[J]. Planta, 2007, 225(6): 1603-1611. DOI: 10.1007/s00425-007-0498-y. 本文引用 [1]

[20]	LIN Y C J, CHEN H, LI Q Z, et al. Reciprocal cross-regulation of VND and SND multigene TF families for wood formation in Populus trichocarpa[J]. PNAS, 2017, 114(45): E9722-E9729. DOI: 10.1073/pnas.1714422114. 本文引用 [1]

[21]	LIU H Z, GAO J H, SUN J T, et al. Dimerization of PtrMYB074 and PtrWRKY19 mediates transcriptional activation of PtrbHLH186 for secondary xylem development in Populus trichocarpa[J]. New Phytololgist, 2022, 234(3): 918-933. DOI: 10.1111/nph.18028. 本文引用 [7]

[22]	SHUAI B, REYNAGA-PEÑA C G, SPRINGER P S. The lateral organ boundaries gene defines a novel, plant-specific gene family[J]. Plant Physiology, 2002, 129(2): 747-761. DOI: 10.1104/pp.010926. 本文引用 [1]

[23]	YAMAGUCHI M, OHTANI M, MITSUDA N, et al. VND-INTERACTING2, a NAC domain transcription factor, negatively regulates xylem vessel formation in Arabidopsis[J]. Plant Cell, 2010, 22(4): 1249-63. DOI: 10.1105/tpc.108.064048. 本文引用 [1]

[24]	YORDANOV Y S, REGAN S, BUSOV V. Members of the lateral organ boundaries domain transcription factor family are involved in the regulation of secondary growth in Populus[J]. Plant Cell, 2010, 22(11): 3662-3677. DOI: 10.1105/tpc.110.078634. 本文引用 [2]

[25]	YU J, ZHOU C G, LI D N, et al. A PtrLBD39-mediated transcriptional network regulates tension wood formation in Populus trichocarpa[J]. Plant Communications, 2021, 3(1): 100250. DOI: 10.1016/j.xplc.2021.100250. 本文引用 [2]

[26]

殷小雨, 胡凤荣, 杨盼盼. LBD转录因子调控植物生长发育的研究进展[J/OL]. 分子植物育种, 2021: 1-11. https: //kns.cnki.net/kcms/detail/46.1068.s.20210708.1419.013.html.

https: //kns.cnki.net/kcms/detail/46.1068.s.20210708.1419.013.html

YIN

X Y

, HU

F R

, YANG

P P

. Research progress of LBD transcription factors regulating plant growth and development[J/OL]. Mol Plant Breed, 2021: 1-11.

本文引用 [1]

[27]	LI S J, ZHEN C, XU W J, et al. Simple, rapid and efficient transformation of genotype Nisqually-1: a basic tool for the first sequenced model tree[J]. Scientific Reports, 2017, 7(1): 2638. DOI: 10.1038/s41598-017-02651-x. 本文引用 [2]

[28]	LIN Y C, LI W, CHEN H, et al. A simple improved-throughput xylem protoplast system for studying wood formation[J]. Nature Protocols, 2014, 9(9): 2194-205. DOI: 10.1038/nprot.2014.147. 本文引用 [2]

[29]	WANG J P, MATTHEWS M L, WILLIAMS C M, et al. Improving wood properties for wood utilization through multi-omics integration in lignin biosynthesis[J]. Nature Communications, 2018, 9(1): 1579. DOI: 10.1038/s41467-018-03863-z. 本文引用 [2]

[30]	LI S, LIN Y C J, WANG P Y, et al. The AREB1 transcription factor influences histone acetylation to regulate drought responses and tolerance in Populus trichocarpa[J]. Plant Cell, 2019, 31(3): 663-686. DOI: 10.1105/tpc.18.00437. 本文引用 [1]

[31]	KASUGA M, LIU Q, MIURA S, et al. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor[J]. Nature Biotechnology, 1999, 17(3): 287-291. DOI: 10.1038/7036. 本文引用 [1]

[32]	YAN Y, WANG P, LU Y, et al. MeRAV5 promotes drought stress resistance in cassava by modulating hydrogen peroxide and lignin accumulation[J]. Plant Journal, 2021, 107(3): 847-860. DOI: 10.1111/tpj.15350. 本文引用 [1]

[33]	康向阳. 林木遗传育种研究进展[J]. 南京林业大学学报(自然科学版), 2020, 44(3): 1-10. KANG X Y. Research progress of forest genetics and tree breeding[J]. Journal of Nanjing Forestry University (Natural Sciences Editon), 2020, 44(3): 1-10. DOI: 10.3969/j.issn.1000-2006.202002033. 本文引用 [1]