转录组分析转录因子AtbHLH68调控细胞壁发育的分子机制

doi:10.13560/j.cnki.biotech.bull.1985.2023-0091

摘要/Abstract

摘要：

细胞壁是植物细胞特有的结构，在参与形态建成、水分和营养物质运输以及抵御生物和非生物胁迫中发挥重要作用。前人研究表明AtbHLH68作为bHLH蛋白的第10亚家族成员，在拟南芥茎维管组织中表达。为探究其在细胞壁发育方面的分子机制，本研究建立了雌二醇诱导pER8-AtbHLH68拟南芥表达系统，实时荧光定量PCR（RT-qPCR）结果显示，10 μmol/L雌二醇处理4 h能够有效诱导AtbHLH68的表达，并且随着诱导时间的增加AtbHLH68的mRNA水平进一步积累，处理8 h后达到对照组29倍。利用转录组测序分析得到了在拟南芥茎中AtbHLH68诱导表达后产生的差异基因。与对照组相比，10 μmol/L雌二醇处理8 h后，共得到差异基因2 334个，其中831个基因显著上调，1 503个基因显著下调。显著富集的GO词条主要与细胞壁组分、防御响应、果胶修饰与降解以及激素响应有关。KEGG分析表明DEGs参与了果胶修饰或木质素生物合成代谢等过程。这些结果表明参与以上过程的差异基因可能被AtbHLH68直接或间接调控，从而导致拟南芥茎细胞壁组分相关基因表达量的变化。该研究为阐明转录因子AtbHLH68在细胞壁发育中的分子机制提供了理论依据。

关键词: 诱导表达, AtbHLH68, 转录组, 细胞壁

Abstract:

Cell wall is a plant-specific structure and plays important role in morphogenesis, water and nutrients transportation, and biotic and abiotic resistance. Previous studies have shown that AtbHLH68 as a member of the 10th bHLH protein subfamily, is expressed in the vascular tissue of Arabidopsis thaliana stem. To investigate its molecular mechanism of AtbHLH68 in cell wall development, the estradiol-induced pER8-AtbHLH68 expression system was established in Arabidopsis thaliana. Quantitative real-time PCR（RT-qPCR）results showed that 10 μmol/L estradiol treatment for 4 h can effectively induce the expression of AtbHLH68, and the expression of AtbHLH68 gradually increased with the prolongation of induction time, reaching 29 times after 8 h. The differentially expressed genes（DEGs）induced by AtbHLH68 in Arabidopsis stems were detected by transcriptome analysis. Compared with the control group, after 8 h of treatment with 10 μmol/L estradiol, a total of 2 334 DEGs were detected, among which 831 genes were significantly up-regulated and 1 503 genes were significantly down-regulated. The significantly enrichment GO terms were mainly related to cell wall components, defense response, pectin modification and degradation and hormone response. KEGG analysis showed that DEGs involved in the processes such as pectin modification and lignin biosynthetic metabolism. These results indicated that the DEGs involved in the above processes may be regulated by AtbHLH68 directly or indirectly. This study provided theoretical evidences to clarify the molecular mechanism of transcription factor AtbHLH68 in cell wall development.

Key words: induced expression, AtbHLH68, transcriptome, cell wall

林红妍, 郭晓蕊, 刘迪, 李慧, 陆海. 转录组分析转录因子AtbHLH68调控细胞壁发育的分子机制[J]. 生物技术通报, 2023, 39(9): 105-116.

LIN Hong-yan, GUO Xiao-rui, LIU Di, LI Hui, LU Hai. Molecular Mechanism of Transcriptional Factor AtbHLH68 in Regulating Cell Wall Development by Transcriptome Analysis[J]. Biotechnology Bulletin, 2023, 39(9): 105-116.

图/表 10

参考文献 37

[1]	Zhang BC, Gao YH, Zhang LJ, et al. The plant cell wall: Biosynthesis, construction, and functions[J]. J Integr Plant Biol, 2021, 63(1): 251-272. doi: 10.1111/jipb.13055
[2]	张保才, 周奕华. 植物细胞壁形成机制的新进展[J]. 中国科学: 生命科学, 2015, 45(6): 544-556.
	Zhang BC, Zhou Y. Plant cell wall formation and regulation[J]. Sci Sin Vitae, 2015, 45(6): 544-556. doi: 10.1360/N052015-00076 URL
[3]	Kumar M, Atanassov I, Turner S. Functional analysis of cellulose synthase(CESA)protein class specificity[J]. Plant Physiol, 2017, 173(2): 970-983. doi: 10.1104/pp.16.01642 URL
[4]	Taylor NG. Cellulose biosynthesis and deposition in higher plants[J]. New Phytol, 2008, 178(2): 239-252. doi: 10.1111/j.1469-8137.2008.02385.x pmid: 18298430
[5]	Scheller HV, Ulvskov P. Hemicelluloses[J]. Annu Rev Plant Biol, 2010, 61: 263-289. doi: 10.1146/annurev-arplant-042809-112315 pmid: 20192742
[6]	陈思慧, 徐华, 黄非, 等. 草本植物半纤维素研究进展[J]. 草业科学, 2020, 37(3): 506-513.
	Chen SH, Xu H, Huang F, et al. Progress in research of grass hemicellulose[J]. Pratacultural Sci, 2020, 37(3): 506-513.
[7]	Vanholme R, Morreel K, Darrah C, et al. Metabolic engineering of novel lignin in biomass crops[J]. New Phytol, 2012, 196(4): 978-1000. doi: 10.1111/j.1469-8137.2012.04337.x pmid: 23035778
[8]	熊媛, 任襄襄, 朱慧, 等. 果胶甲酯酶抑制蛋白(PMEIs)的功能性研究进展[J]. 中国生物工程杂志, 2022, 42(8): 99-108.
	Xiong Y, Ren XX, Zhu H, et al. Recent progress in the functional research of PMEIs[J]. China Biotechnol, 2022, 42(8): 99-108.
[9]	Willats WGT, Orfila C, Limberg G, et al. Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls[J]. J Biol Chem, 2001, 276(22): 19404-19413. doi: 10.1074/jbc.M011242200 pmid: 11278866
[10]	张海燕, 吴天祥. 微生物果胶酶的研究进展[J]. 酿酒科技, 2006(9): 82-85.
	Zhang HY, Wu TX. Research progress in pectinase[J]. Liquor Mak Sci Technol, 2006(9): 82-85.
[11]	陈乐天, 王慧婷, 韩靖鸾, 等. 植物果胶裂解酶的研究现状及展望[J]. 华南农业大学学报, 2019, 40(5): 71-77.
	Chen LT, Wang HT, Han JL, et al. Research progress and perspective of plant pectin lysase[J]. J South China Agric Univ, 2019, 40(5): 71-77.
[12]	Terrett OM, Dupree P. Covalent interactions between lignin and hemicelluloses in plant secondary cell walls[J]. Curr Opin Biotechnol, 2019, 56: 97-104. doi: 10.1016/j.copbio.2018.10.010 URL
[13]	余紫苹, 彭红, 林妲, 等. 植物半纤维素结构研究进展[J]. 高分子通报, 2011(6): 48-54.
	Yu ZP, Peng H, Lin D, et al. Research progress of plant hemicellulose structure[J]. Polym Bull, 2011(6): 48-54.
[14]	刘佩佩, 张耿, 李晓娟. 植物果胶的生物合成与功能[J]. 植物学报, 2021, 56(2): 191-200.
	Liu PP, Zhang G, Li XJ. Biosynthesis and function of plant pectin[J]. Chin Bull Bot, 2021, 56(2): 191-200.
[15]	Cosgrove DJ. Growth of the plant cell wall[J]. Nat Rev Mol Cell Biol, 2005, 6(11): 850-861.
[16]	Li Z, Fernie AR, Persson S. Transition of primary to secondary cell wall synthesis[J]. Sci Bull, 2016, 61(11): 838-846. doi: 10.1007/s11434-016-1061-7 URL
[17]	Heim MA, Jakoby M, Werber M, et al. The basic Helix-loop-Helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity[J]. Mol Biol Evol, 2003, 20(5): 735-747. doi: 10.1093/molbev/msg088 URL
[18]	Gao ZY, Sun WJ, Wang J, et al. GhbHLH18 negatively regulates fiber strength and length by enhancing lignin biosynthesis in cotton fibers[J]. Plant Sci, 2019, 286: 7-16. doi: S0168-9452(19)30265-1 pmid: 31300144
[19]	Zhao WQ, Ding L, Liu JY, et al. Regulation of lignin biosynthesis by an atypical bHLH protein CmHLB in Chrysanthemum[J]. J Exp Bot, 2022, 73(8): 2403-2419. doi: 10.1093/jxb/erac015 URL
[20]	Yan L, Xu CH, Kang YL, et al. The heterologous expression in Arabidopsis thaliana of sorghum transcription factor SbbHLH1 downregulates lignin synthesis[J]. J Exp Bot, 2013, 64(10): 3021-3032. doi: 10.1093/jxb/ert150 pmid: 23698629
[21]	Cao YY, Zeng HX, Ku LX, et al. ZmIBH1-1 regulates plant architecture in maize[J]. J Exp Bot, 2020, 71(10): 2943-2955. doi: 10.1093/jxb/eraa052 pmid: 31990030
[22]	Le Hir R, Castelain M, Chakraborti D, et al. AtbHLH68 transcription factor contributes to the regulation of ABA homeostasis and drought stress tolerance in Arabidopsis thaliana[J]. Physiol Plantarum, 2017, 160(3): 312-327. doi: 10.1111/ppl.2017.160.issue-3 URL
[23]	Ikeda M, Fujiwara S, Mitsuda N, et al. A triantagonistic basic helix-loop-helix system regulates cell elongation in Arabidopsis[J]. Plant Cell, 2012, 24(11): 4483-4497. doi: 10.1105/tpc.112.105023 URL
[24]	Oh J, Park E, Song K, et al. PHYTOCHROME INTERACTING FACTOR8 inhibits phytochrome A-mediated far-red light responses in Arabidopsis[J]. Plant Cell, 2020, 32(1): 186-205. doi: 10.1105/tpc.19.00515 URL
[25]	Liu WW, Tai HH, Li SS, et al. bHLH122 is important for drought and osmotic stress resistance in Arabidopsis and in the repression of ABA catabolism[J]. New Phytol, 2014, 201(4): 1192-1204. doi: 10.1111/nph.2014.201.issue-4 URL
[26]	Xu WJ, Dubos C, Lepiniec L. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes[J]. Trends Plant Sci, 2015, 20(3): 176-185. doi: 10.1016/j.tplants.2014.12.001 pmid: 25577424
[27]	Coculo D, Lionetti V. The plant invertase/pectin methylesterase inhibitor superfamily[J]. Front Plant Sci, 2022, 13: 863892. doi: 10.3389/fpls.2022.863892 URL
[28]	Guo XQ, Chang S, Hu JP, et al. Research progress of pectin methylesterase and its inhibitors[J]. Curr Protein Pept Sci, 2022, 23(10): 684-696. doi: 10.2174/1389203723666220919092428 URL
[29]	Shi DC, Ren AY, Tang XF, et al. MYB52 negatively regulates pectin demethylesterification in seed coat mucilage[J]. Plant Physiol, 2018, 176(4): 2737-2749. doi: 10.1104/pp.17.01771 URL
[30]	Wilson MH, Holman TJ, SÃ¸rensen I, et al. Multi-omics analysis identifies genes mediating the extension of cell walls in the Arabidopsis thaliana root elongation zone[J]. Front Cell Dev Biol, 2015, 3: 10.
[31]	Han Y, Ban QY, Hou YL, et al. Isolation and characterization of two persimmon xyloglucan endotransglycosylase/hydrolase(XTH)genes that have divergent functions in cell wall modification and fruit postharvest softening[J]. Front Plant Sci, 2016, 7: 624. doi: 10.3389/fpls.2016.00624 pmid: 27242828
[32]	Daniel B, Pavkov-Keller T, Steiner B, et al. Oxidation of monolignols by members of the berberine bridge enzyme family suggests a role in plant cell wall metabolism[J]. J Biol Chem, 2015, 290(30): 18770-18781. doi: 10.1074/jbc.M115.659631 pmid: 26037923
[33]	Fernández-Pérez F, Pomar F, Pedreño MA, et al. The suppression of AtPrx52 affects fibers but not xylem lignification in Arabidopsis by altering the proportion of syringyl units[J]. Physiol Plantarum, 2015, 154(3): 395-406. doi: 10.1111/ppl.2015.154.issue-3 URL
[34]	Kärkönen A, Fry SC. Effect of ascorbate and its oxidation products on H₂O₂ production in cell-suspension cultures of Picea abies and in the absence of cells[J]. J Exp Bot, 2006, 57(8): 1633-1644. doi: 10.1093/jxb/erj197 pmid: 16698813
[35]	Hamann T. The plant cell wall integrity maintenance mechanism-A case study of a cell wall plasma membrane signaling network[J]. Phytochemistry, 2015, 112: 100-109. doi: 10.1016/j.phytochem.2014.09.019 URL
[36]	Zhang NL, Zhou S, Yang DY, et al. Revealing shared and distinct genes responding to JA and SA signaling in Arabidopsis by meta-analysis[J]. Front Plant Sci, 2020, 11: 908. doi: 10.3389/fpls.2020.00908 URL
[37]	Chen JN, Nolan T, Ye HX, et al. Arabidopsis WRKY46, WRKY54 and WRKY70 transcription factors are involved in brassinosteroid-regulated plant growth and drought response[J]. Plant Cell, 2017: tpc. 00364.2017.

基因ID Unigene ID	引物名称 Primer name	引物序列 Primer sequence（5'-3'）
AT3G18780	Actin2-F	CGTATGAGCAAGGAGATCAC
AT3G18780	Actin2-R	CACATCTGTTGGAAGGTGCT
AT4G29100	bHLH68-F	ACCACGGATGATGCCCACT
AT4G29100	bHLH68-R	CCATCATCAATCCACCCAAAA
AT1G56100	PMEI-F	TGGTTGCCTCGTTGGATG
AT1G56100	PMEI-R	TGTGTAGGTTGACCATTGATGTAAA
AT2G47030	VGD1-F	GCACAGTCCAGGTTGAGTCCGAAGG
AT2G47030	VGD1-R	GGTCTCCGTTCACTCTGATAGCCAC
AT4G34230	CAD5-F	GTTGCCTCGTTGGATGTTGC
AT4G34230	CAD5-R	CGCCTTGTGTAGGTTGACCATTGAT
AT5G62350	PMEI-F	ACTAAGCCGAGCCCAATCC
AT5G62350	PMEI-R	GTATCGTTCATCTCCTCGACGCAAT
AT3G56400	WRKY70-F	GCGATAGTCGGAAAAGATTGG
AT3G56400	WRKY70-R	TGGGAATTTGGCATTAAGAA
AT1G30760	BBE13-F	TCGGCGTAATACTTGCCTGGAAAAT
AT1G30760	BBE13-R	TATCAGCGACTTGTTGCCACTT
	YXM-pER8-bHLH68-F	GGTAATGCCATGTAATATGCTCG
	YXM-pER8-bHLH68-R	CATCATCAATCCACCCAAAA

基因ID Unigene ID	引物名称 Primer name	引物序列 Primer sequence（5'-3'）
AT3G18780	Actin2-F	CGTATGAGCAAGGAGATCAC
AT3G18780	Actin2-R	CACATCTGTTGGAAGGTGCT
AT4G29100	bHLH68-F	ACCACGGATGATGCCCACT
AT4G29100	bHLH68-R	CCATCATCAATCCACCCAAAA
AT1G56100	PMEI-F	TGGTTGCCTCGTTGGATG
AT1G56100	PMEI-R	TGTGTAGGTTGACCATTGATGTAAA
AT2G47030	VGD1-F	GCACAGTCCAGGTTGAGTCCGAAGG
AT2G47030	VGD1-R	GGTCTCCGTTCACTCTGATAGCCAC
AT4G34230	CAD5-F	GTTGCCTCGTTGGATGTTGC
AT4G34230	CAD5-R	CGCCTTGTGTAGGTTGACCATTGAT
AT5G62350	PMEI-F	ACTAAGCCGAGCCCAATCC
AT5G62350	PMEI-R	GTATCGTTCATCTCCTCGACGCAAT
AT3G56400	WRKY70-F	GCGATAGTCGGAAAAGATTGG
AT3G56400	WRKY70-R	TGGGAATTTGGCATTAAGAA
AT1G30760	BBE13-F	TCGGCGTAATACTTGCCTGGAAAAT
AT1G30760	BBE13-R	TATCAGCGACTTGTTGCCACTT
	YXM-pER8-bHLH68-F	GGTAATGCCATGTAATATGCTCG
	YXM-pER8-bHLH68-R	CATCATCAATCCACCCAAAA

样品名 Sample	原始序列数 Raw reads/M	高质量序列数 Clean reads/M	Q20/%	Q30/%	比对到参考基因组序列数 No. of alignment reference genome sequences/%	唯一参考序列数 No. of unique alignment sequences/%
4 h	47.33	45.22	98.02	94.51	96.83	95.08
6 h	45.57	43.95	97.83	93.99	97.12	95.35
8 h	45.57	44.01	97.78	93.86	96.74	94.97
Control	47.33	45	97.9	94.18	96.13	94.11

样品名 Sample	原始序列数 Raw reads/M	高质量序列数 Clean reads/M	Q20/%	Q30/%	比对到参考基因组序列数 No. of alignment reference genome sequences/%	唯一参考序列数 No. of unique alignment sequences/%
4 h	47.33	45.22	98.02	94.51	96.83	95.08
6 h	45.57	43.95	97.83	93.99	97.12	95.35
8 h	45.57	44.01	97.78	93.86	96.74	94.97
Control	47.33	45	97.9	94.18	96.13	94.11

基因表达Gene expression	基因号Gene ID	Log₂x	P值P Value	描述Description
下调基因	AT2G06850	-1.06394221	1.85E-50	Xyloglucan endotransglucosylase/hydrolase 4
Down-regulated gene	AT2G36870	-1.266714752	3.34E-19	Xyloglucan endotransglucosylase/hydrolase 32
	AT4G25820	-2.74819285	1.28E-06	Xyloglucan endotransglucosylase/hydrolase 14
	AT5G57530	-5.654911378	8.25E-28	Xyloglucan endotransglucosylase/hydrolase 12
	AT5G57540	-3.641717279	7.07E-17	Xyloglucan endotransglucosylase/hydrolase 13
	AT1G71695	-1.298988679	3.66E-22	Peroxidase superfamily protein
	AT1G30870	-3.50779464	6.16E-08	Peroxidase 7
	AT3G49110	-2.420843121	6.80E-11	Peroxidase 33
	AT3G50990	-2.306246473	8.33E-06	Peroxidase 36
	AT4G37980	-1.756033681	3.73E-18	Cinnamyl-alcohol dehydrogenase 7
	AT2G47030	-2.29336778	2.45E-142	Pectin methylesterase 4
	AT2G47040	-2.786313785	0	Vanguard1, VGD1
	AT3G10720	-2.090197809	2.92E-13	Pectin methylesterase 25
	AT3G62170	-3.528498693	4.35E-207	Pectin methylesterase 37
	AT4G15980	-2.788495895	1.82E-05	Pectin methylesterase 43
	AT5G07410	-4.717215431	2.64E-240	Pectin methylesterase 48
	AT3G05610	-2.626956589	1.03E-120	Pectin methylesterase 21
	AT2G26440	-2.370550991	3.78E-132	Pectin methylesterase 12
	AT3G06830	-2.258402957	2.42E-15	Plant invertase/pectin methylesterase inhibitor superfamily
	AT5G07420	-4.837943242	2.77E-08	Pectin lyase-like superfamily protein
	AT5G07430	-2.606610505	7.12E-169	Pectin lyase-like superfamily protein
	AT3G17060	-2.165538573	6.39E-22	Pectin lyase-like superfamily protein
	AT1G14420	-2.298373367	3.91E-46	Pectin lyase-like superfamily protein
	AT2G02720	-2.19730144	4.49E-24	Pectin lyase-like superfamily protein
	AT3G01270	-2.71241092	0	Pectin lyase-like superfamily protein
	AT5G15110	-2.336194664	2.35E-29	Pectate lyase family protein
	AT5G48900	-6.312882955	1.10E-22	Pectin lyase-like superfamily protein
	AT5G55720	-3.135159583	4.76E-11	Pectin lyase-like superfamily protein
	AT3G45860	-1.803254412	3.34E-18	Cysteine-rich RLK（receptor-like protein kinase）4
	AT4G23170	-1.533408039	1.62E-109	Cysteine-rich RLK（receptor-like protein kinase）9
	AT2G40750	-1.152515323	8.63E-64	WRKY DNA-binding protein 54
	AT2G46400	-1.299919474	3.63E-61	WRKY DNA-binding protein 46
	AT3G56400	-1.40629482	0	WRKY DNA-binding protein 70
	AT4G23810	-1.452162691	1.12E-65	WRKY DNA-binding protein 53
	AT5G24780	-1.294335074	0	Vegetative storage protein 1
	AT3G16470	-1.212665758	0	A JA-responsive gene that coordinates with GRP 7
	AT2G32690	-2.181267688	6.01E-32	Glycine-rich protein 23
	AT1G20510	-1.035196623	4.70E-93	OPC-8:CoA ligase involved in jasmonic acid biosynthesis
上调基因	AT1G11545	2.054076673	1.34E-06	Xyloglucan endotransglucosylase/hydrolase 8
Up-regulated gene	AT3G48580	3.262777493	3.34E-61	Xyloglucan endotransglucosylase/hydrolase 11
	AT4G14130	1.838579331	4.09E-18	Xyloglucan endotransglucosylase/hydrolase 15
	AT4G37800	2.408582477	8.79E-21	Xyloglucan endotransglucosylase/hydrolase 7
	AT1G30700	3.103198888	3.07E-254	FAD-binding berberine family protein, AtBBE 7
	AT1G30720	3.04026387	2.09E-22	FAD-binding berberine family protein, AtBBE 10
	AT1G30730	2.767165832	8.28E-09	FAD-binding berberine family protein, AtBBE 11
	AT4G20800	3.940837928	8.47E-14	FAD-binding berberine family protein, AtBBE 17
	AT5G44390	2.190683562	4.52E-05	FAD-binding berberine family protein, AtBBE 25
	AT5G44400	2.779652634	0	FAD-binding berberine family protein, AtBBE 26
	AT1G30760	1.250535773	3.26e-210	Berberine bridge enzyme-like 13, AtBBE 13
	AT2G34790	2.036462643	1.06E-182	Berberine bridge enzyme-like 15, AtBBE 15
	AT4G36430	2.677849798	3.42E-36	Peroxidase superfamily protein
	AT3G21770	1.766366511	5.33E-07	Peroxidase superfamily protein
	AT3G28200	1.80094768	1.06E-34	Peroxidase superfamily protein
	AT1G05260	1.600816452	2.62E-31	Peroxidase 3
	AT5G05340	2.40141963	1.48E-80	Peroxidase 52
	AT5G60020	1.132349101	5.96E-135	Laccase 17
	AT4G34230	1.168213629	0	Cinnamyl alcohol dehydrogenase 5
	AT4G37990	1.401205226	3.90E-06	Cinnamyl-alcohol dehydrogenase 8
	AT1G11590	2.950611973	1.48E-19	Pectin methylesterase 19
	AT5G09760	1.08363098	2.08E-22	Pectin methylesterase 15
	AT1G14890	1.984238146	0	Plant invertase/pectin methylesterase inhibitor superfamily protein
	AT5G62350	1.901654358	0	Plant invertase/pectin methylesterase inhibitor superfamily protein
	AT1G62770	1.620506618	3.20E-05	Pectin methyleseterase inhibitor 19
	AT1G56100	1.25	0	Plant invertase/pectin methylesterase inhibitor 14
	AT3G47380	1.787395021	2.75E-23	Plant invertase/pectin methylesterase inhibitor 11
	AT3G27400	2.985665084	5.10E-106	Pectin lyase-like superfamily protein
	AT3G53190	1.145912382	1.86E-14	Pectin lyase-like superfamily protein
	AT4G13710	1.186011986	2.50E-09	Pectin lyase-like superfamily protein
	AT4G24780	1.1622914	1.26E-84	Pectate lyase 19
	AT5G04310	1.318331844	6.35E-11	Pectate lyase like 12