林业科学 ›› 2021, Vol. 57 ›› Issue (9): 42-51.doi: 10.11707/j.1001-7488.20210905
周紫晶1,范付华1,*,尚先文1,覃慧娟1,王聪慧1,丁贵杰1,谭健晖2
收稿日期:
2021-04-21
出版日期:
2021-09-25
发布日期:
2021-11-29
通讯作者:
范付华
基金资助:
Zijing Zhou1,Fuhua Fan1,*,Xianwen Shang1,Huijuan Qin1,Conghui Wang1,Guijie Ding1,Jianhui Tan2
Received:
2021-04-21
Online:
2021-09-25
Published:
2021-11-29
Contact:
Fuhua Fan
摘要:
目的: 对2年生马尾松幼苗进行不同浓度IAA喷施处理,结合生理生化、形态解剖结构及转录组水平变化等综合分析,揭示外源IAA对马尾松苗期茎干次生生长的影响机制,为阐明马尾松苗期茎干次生生长的分子调控机制奠定基础,可以为培育速生高产的马尾松新品系提供一定的理论参考。方法: 对2年生马尾松幼苗进行不同浓度IAA(0、1、50、100 mg·L-1)喷施处理,处理160天后进行生长指标、解剖结构、生理生化指标的测定;通过高通量转录组测序鉴定差异表达基因,对差异表达基因进行GO和KEGG功能分析。结果: IAA处理对2年生马尾松的地径、细胞生长(木质部、韧皮部厚度以及木质部细胞层数)、木材主要成分(木质素、纤维素及半纤维素含量)及内源激素含量(生长素、赤霉素及油菜素内酯)具有显著的促进作用(P<0.05);通过Illumina测序技术对对照组和IAA处理的6个cDNA文库进行测序,最终得到IAA处理后差异表达基因778个(包括482个上调表达基因和296个下调表达基因),GO分析表明这些差异基因主要富集于细胞成分、分子功能和生物过程中共计22个类别,KEGG分析表明它们参与了64种不同的途径,并筛选出与马尾松次生生长相关的差异表达基因(BRL1、GASA6、CAD和CESA2等)。结论: IAA处理能够改变内源激素含量,促进细胞分裂,增加木质素、纤维素和半纤维素积累,从而促进马尾松茎干的次生生长。
中图分类号:
周紫晶,范付华,尚先文,覃慧娟,王聪慧,丁贵杰,谭健晖. 外源IAA对马尾松幼苗茎干次生生长的影响[J]. 林业科学, 2021, 57(9): 42-51.
Zijing Zhou,Fuhua Fan,Xianwen Shang,Huijuan Qin,Conghui Wang,Guijie Ding,Jianhui Tan. Effects of Exogenous IAA on Stem Secondary Growth of Pinus massoniana Seedlings[J]. Scientia Silvae Sinicae, 2021, 57(9): 42-51.
表1
引物序列"
基因 Gene | 序列 Sequence(5′→3′) |
TRINITY_DN10179_c0_g1 | F: TGATGGATGGTGGTTGATAC |
R: CTGATGGGCAAACTGAGATA | |
TRINITY_DN8564_c0_g2 | F: GCGTATTCCGCCAGAGTG |
R: TCCAGAACGAGTGCCCCT | |
TRINITY_DN5190_c1_g1 | F: CAGGCGACTCCACCTACAACT |
R: TGCGGAACACCTAATCCAAAC | |
TRINITY_DN2065_c0_g1 | F: GAACGCAGAGCAATGAAG |
R: AAGCCCACCACTATGACC | |
TRINITY_DN11133_c0_g2 | F: TCACGCTTCCTTTGGCTTTT |
R: CGTACGGTCTGCACTTCATC | |
TRINITY_DN3030_c1_g1 | F: AGAGCAGATTCCAGCAGA |
R: CGGGTTTCCAGTAGATGA | |
TRINITY_DN39456_c0_g1 | F: TCAGCGGACCCTGTTCTA |
R: GACAAAGTTGCCGTGGAG | |
TRINITY_DN40433_c0_g1 | F: AGGGACTGGCTCCTTGTG |
R: CTTCGGAATCGCACCTTT | |
TRINITY_DN99422_c0_g3 | F: GAAGACTTGAAATGCCTGAC |
R: ATAGACCTTGAACGCCTC | |
UBC | F: GATTTATTTCATTGGCAGGC |
R: AGGATCATCAGGATTTGGGT |
表2
IAA处理后马尾松幼苗茎干的木质素、纤维素和半纤维含量"
IAA | 木质素含量 Lignin content/(mg·g-1) | 纤维素含量 Cellulose content/(mg·g-1) | 半纤维素含量 Hemicellulose content/(mg·g-1) |
CK | 89.95±0.98 a | 199.30±35.81 a | 160.51±1.01 a |
1 mg·L-1 | 116.33±0.07 b | 292.35±18.03 b | 207.13±15.40 b |
50 mg·L-1 | 122.43±3.92 b | 287.18±16.06 b | 202.68±5.75 b |
100 mg·L-1 | 124.94±7.08 b | 285.85±12.47 b | 187.36±3.05 ab |
表3
IAA处理后马尾松幼苗茎干内源激素含量①"
内源激素Endogenous hormone/(ng·g-1) | CK | IAA处理 IAA treatment | |
生长素类 Auxins | 吲哚-3-乙酸Indole-3-acetic acid(IAA) | 58.67±1.95 | 113.67±4.91** |
氧化吲哚乙酸Oxidized indoleacetic acid(OxIAA) | 0.00 | 114.00±4.73** | |
吲哚-3-甲醛Indole-3-carboxaldehyde(ICAld) | 4.48±0.04 | 133.00±3.51** | |
吲哚-3-乙酸甲酯Indole-3-methyl acetate(MEIAA) | 3.85±0.10 | 7.18±0.28** | |
吲哚乙酸-亮氨酸Indoleacetic acid-leucine(IAA-Leu) | 1.06±0.08** | 0.56±0.03 | |
吲哚乙酸-天冬氨酸Indoleacetic acid-aspartic acid(IAA-Asp) | 1.89±0.13** | 1.27±0.06 | |
色胺Tryptamine(TRA) | 0.16±0.01 | 0.14±0.00 | |
色氨酸Tryptophan(TRP) | 2 156.67±53.64* | 1 903.33±43.33 | |
3-吲哚丙酸3-Indolepropionic acid(IPA) | 42.63±0.92 | 44.30±2.86 | |
赤霉素类 Gibberellins | 赤霉素1 Gibberellin1(GA1) | 17.67±0.69 | 24.60±2.46 |
赤霉素15 Gibberellin15(GA15) | 0.27±0.05 | 0.27±0.03 | |
油菜素内酯类 Brassinolides | 油菜素内酯Brassinolide(BR) | 0.05±0.00 | 0.09±0.00** |
油菜素甾酮Castasterone(CS) | 19.89±1.18 | 27.03±0.99** | |
28-高油菜素甾酮28-homocastasterone(28-homoCS) | 1.25±0.07 | 1.58±0.03** | |
香蒲甾醇Typhasterol(TY) | 36.38±0.31 | 35.42±0.32 | |
6-脱氧油菜素甾酮6-deoxocastasterone(6-deoxoCS) | 0.02±0.00 | 0.03±0.00 |
表4
IAA处理后马尾松幼苗茎部转录组数据统计①"
样本 Sample | 原始读数 Raw reads | 清洁读数(清洁读数/原始读数) Clean reads(clean reads/raw reads) | 错误率 Error rate(%) | Q20(%) | Q30(%) | GC含量 GC content(%) |
CK1 | 79 111 552 | 78 288 654(98.96%) | 0.024 4 | 98.31 | 94.73 | 44.47 |
CK2 | 73 641 850 | 72 846 758(98.92%) | 0.024 3 | 98.33 | 94.83 | 44.96 |
CK3 | 86 263 202 | 85 424 670(99.03%) | 0.024 4 | 98.28 | 94.66 | 44.65 |
IAA1 | 87 975 948 | 87 084 030(98.99%) | 0.024 2 | 98.36 | 94.87 | 44.83 |
IAA2 | 93 638 284 | 92 535 608(98.82%) | 0.024 6 | 98.24 | 94.55 | 45.16 |
IAA3 | 77 429 912 | 76 588 978(98.91%) | 0.024 7 | 98.17 | 94.39 | 44.87 |
表5
次生生长相关基因的鉴定与表达"
基因号 Gene ID | 注释 Description | 差异倍数 Log2FC | 表达量Expression quantity | |
CK | IAA | |||
激素Hormone | ||||
TRINITY_DN6209_c0_g1 | 油菜素内酯受体激酶Brassinosteroid LRR receptor kinase(BRL1) | 1.92 | 9.15 | 30.11 |
TRINITY_DN105600_c0_g2 | 赤霉素响应蛋白Gibberellin-regulated protein(GASA6) | 1.70 | 2.09 | 6.35 |
木质素Lignin | ||||
TRINITY_DN40433_c0_g1 | 肉桂醇脱氢酶Cinnamyl alcohol dehydrogenases(CAD) | 7.48 | 0 | 1.06 |
纤维素Cellulose | ||||
TRINITY_DN2065_c0_g1 | 纤维素合酶Cellulose synthase(CESA2) | 1.13 | 23.30 | 45.01 |
陈海燕, 刘凯, 余敏, 等. 外源激素IAA和GA3对马尾松应压木形成的影响. 北京林业大学学报, 2015, 37 (5): 134- 139. | |
Chen H Y , Liu K , Yu M , et al. Effect of exogenous IAA and GA3 on formation of compression wood of Pinus massoniana Lamb. Journal of Beijing Forestry University, 2015, 37 (5): 134- 139. | |
丁贵杰, 严仁发, 齐新民. 不同种源马尾松造林效果及经济效益对比分析. 林业科学, 1994, (6): 506- 512. | |
Ding G J , Yan R F , Qi X M . A comparative analysis on planting result and economic benefit of Pinus massoniana Lamb. from different provenance. Scientia Silvae Sinicae, 1994, (6): 506- 512. | |
郭丽玲, 潘萍, 欧阳勋志, 等. 间伐补植对马尾松低效林生长及林分碳密度的短期影响. 西南林业大学学报: 自然科学, 2019, 39 (3): 48- 54. | |
Guo L L , Pan P , Ouyang X Z , et al. Short-term effect of thinning and replanting measures on tree growth and stand carbon density of low-efficiency Pinus massoniana forest. Journal of Southwest Forestry University: Natural Science, 2019, 39 (3): 48- 54. | |
吴修蓉, 周运超, 余星. 镁肥对马尾松幼苗生长与叶片元素积累的影响. 亚热带植物科学, 2019, 48 (1): 11- 16. | |
Wu X R , Zhou Y C , Yu X . Effect of magnesium fertilizer on growth and element accumulation of Pinus massoniana seedlings. Subtropical Plant Science, 2019, 48 (1): 11- 16. | |
张婷, 文晓鹏. 混合接种外生菌根菌对马尾松种子萌发及幼苗生长的影响. 种子, 2016, 35 (10): 1- 5.
doi: 10.3969/j.issn.1000-8071.2016.10.001 |
|
Zhang T , Wen X P . Effect of ectomycorrhizal fungi combination on seed germination and seedling growth of Masson Pine(Pinus massoniana). Seed, 2016, 35 (10): 1- 5.
doi: 10.3969/j.issn.1000-8071.2016.10.001 |
|
周乃富, 张俊佩, 刘昊, 等. 木本植物非均质化组织石蜡切片制作方法. 植物学报, 2018, 53 (5): 653- 660. | |
Zhou N F , Zhang J P , Liu H , et al. New protocols for paraffin sections of heterogeneous tissues of woody plants. Chinese Bulletin of Botany, 2018, 53 (5): 653- 660. | |
Bargmann B O R , Vanneste S , Krouk G , et al. A map of cell type-specific auxin responses. Molecular Systems Biology, 2013, 9, 688.
doi: 10.1038/msb.2013.40 |
|
Ben-Nissan G , Lee J , Borohov A , et al. GIP, a Petunia hybrida GA-induced cysteine-rich protein: a possible role in shoot elongation and transition to flowering. The Plant Journal, 2004, 37 (2): 229- 238.
doi: 10.1046/j.1365-313X.2003.01950.x |
|
Bjorklund S , Antti H , Uddestrand I , et al. Cross-talk between gibberellin and auxin in development of Populus wood: gibberellin stimulates polar auxin transport and has a common transcriptome with auxin. The Plant Journal, 2007, 52 (3): 499- 511.
doi: 10.1111/j.1365-313X.2007.03250.x |
|
Brackmann K , Qi J , Gebert M , et al. Spatial specificity of auxin responses coordinates wood formation. Nature Communications, 2018, 9, 875.
doi: 10.1038/s41467-018-03256-2 |
|
Cano-Delgado A , Yin Y , Yu C , et al. BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis. Development, 2004, 131 (21): 5341- 5351.
doi: 10.1242/dev.01403 |
|
Chaiwanon J , Wang Z . Spatiotemporal brassinosteroid signaling and antagonism with auxin pattern stem cell dynamics in Arabidopsis roots. Current Biology, 2015, 25 (8): 1031- 1042.
doi: 10.1016/j.cub.2015.02.046 |
|
Chen J , Wang L , Immanen J , et al. Differential regulation of auxin and cytokinin during the secondary vascular tissue regeneration in Populus trees. New Phytologist, 2019, 224 (1): 188- 201.
doi: 10.1111/nph.16019 |
|
Fan F, Cui B, Zhang T, et al. 2014. The temporal transcriptomic response of Pinus massoniana seedlings to phosphorus deficiency. PLoS ONE, 9: e1050688. | |
Glweiler L , Uan C , Ller A , et al. Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissues. Science, 1998, 282 (5397): 2226- 2230.
doi: 10.1126/science.282.5397.2226 |
|
Joshi C P , Bhandari S , Ranjan P , et al. Genomics of cellulose biosynthesis in poplars. New Phytology, 2004, 164, 53- 61.
doi: 10.1111/j.1469-8137.2004.01155.x |
|
Li A , Xia T , Xu W , et al. An integrative analysis of four CESA isoforms specific for fiber cellulose production between Gossypium hirsutum and Gossypium barbadense. Planta, 2013, 237 (6): 1585- 1597.
doi: 10.1007/s00425-013-1868-2 |
|
Livak K J , Schmittgen T D . Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method. Methods, 2001, 25 (4): 402- 408.
doi: 10.1006/meth.2001.1262 |
|
Ma D , Xu C , Alejos-Gonzalez F , et al. Overexpression of Artemisia annua cinnamyl alcohol dehydrogenase increases lignin and coumarin and reduces artemisinin and other sesquiterpenes. Frontiers in Plant Science, 2018, 9, 828.
doi: 10.3389/fpls.2018.00828 |
|
Maleki S S , Mohammadi K , Ji K . Characterization of cellulose synthesis in plant cells. The Scientific World Journal, 2016, 2016, 8641373. | |
Maleki S S , Mohammadi K , Movahedi A , et al. Increase in cell wall thickening and biomass production by overexpression of PmCesA2 in poplar. Frontiers in Plant Science, 2020, 11, 110.
doi: 10.3389/fpls.2020.00110 |
|
Mellerowicz E J , Baucher M , Sundberg B , et al. Unravelling cell wall formation in the woody dicot stem. Plant Molecular Biology, 2001, 47 (1/2): 239- 274.
doi: 10.1023/A:1010699919325 |
|
Muller C J , Valdes A E , Wang G , et al. PHABULOSA mediates an auxin signaling loop to regulate vascular patterning in Arabidopsis. Plant Physiology, 2016, 170 (2): 956- 970.
doi: 10.1104/pp.15.01204 |
|
Nairn C J , Haselkorn T . Three loblolly pine CesA genes expressed in developing xylem are orthologous to secondary cell wall CesA genes of angiosperms. The New Phytologist, 2005, 166 (3): 907- 915.
doi: 10.1111/j.1469-8137.2005.01372.x |
|
Nemhauser J L , Mockler T C , Chory J . Interdependency of brassinosteroid and auxin signaling in Arabidopsis. PLoS Biology, 2004, 2 (9): E258.
doi: 10.1371/journal.pbio.0020258 |
|
Nieminen K M , Kauppinen L , Helariutta Y . A weed for wood? Arabidopsis as a genetic model for xylem development. Plant Physiology, 2004, 135 (2): 653- 659.
doi: 10.1104/pp.104.040212 |
|
Pan H , Zhou R , Louie G V , et al. Structural studies of cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase, key enzymes of monolignol biosynthesis. Plant Cell, 2014, 26 (9): 3709- 3727.
doi: 10.1105/tpc.114.127399 |
|
Pan X , Welti R , Wang X . Quantitative analysis of major plant hormones in crude plant extracts by high-performance liquid chromatography-mass spectrometry. Nature Protocols, 2010, 5 (6): 986- 992.
doi: 10.1038/nprot.2010.37 |
|
Ponniah S K , Shang Z , Akbudak M A , et al. Down-regulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase, cinnamoyl CoA reductase, and cinnamyl alcohol dehydrogenase leads to lignin reduction in rice(Oryza sativa L. ssp. japonica cv. Nipponbare). Plant Biotechnology Reports, 2017, 11 (1): 17- 27. | |
Sakamoto T , Morinaka Y , Inukai Y , et al. Auxin signal transcription factor regulates expression of the brassinosteroid receptor gene in rice. Plant Journal, 2013, 73 (4): 676- 688.
doi: 10.1111/tpj.12071 |
|
Savidge R A . Auxin and ethylene regulation of diameter growth in trees. Tree Physiology, 1988, 4 (4): 401- 414.
doi: 10.1093/treephys/4.4.401 |
|
Savidge R A . The role of plant hormones in higher plant cellular differentiation Ⅱ: Experiments with the vascular cambium, and sclereid and tracheid differentiation in the pine, Pinus contorta. The Histochemical Journal, 1983, 15 (5): 447- 466.
doi: 10.1007/BF01002699 |
|
Spicer R , Groover A . Evolution of development of vascular cambia and secondary growth. New Phytologist, 2010, 186 (3): 577- 592.
doi: 10.1111/j.1469-8137.2010.03236.x |
|
Taylor B H , Scheuring C F . A molecular marker for lateral root initiation: the RSI-1 gene of tomato(Lycopersicon esculentum Mill) is activated in early lateral root primordia. Molecular General Genetics, 1994, 243 (2): 148- 157.
doi: 10.1007/BF00280311 |
|
Tuominen H , Puech L , Fink S , et al. A radial concentration gradient of indole-3-acetic acid is related to secondary xylem development in hybrid aspen. Plant Physiology, 1997, 115 (2): 577- 585.
doi: 10.1104/pp.115.2.577 |
|
Uggla C , Mellerowicz E J , Sundberg B . Indole-3-acetic acid controls cambial growth in Scots pine by positional signaling. Plant Physiology, 1998, 117 (1): 113- 121.
doi: 10.1104/pp.117.1.113 |
|
Yao X , Remko O . PDK1 regulates auxin transport and Arabidopsis vascular development through AGC1 kinase PAX. Nature Plants, 2020, 6, 544- 555.
doi: 10.1038/s41477-020-0650-2 |
|
Yu M , Liu K , Liu S , et al. Effect of exogenous IAA on tension wood formation by facilitating polar auxin transport and cellulose biosynthesis in hybrid poplar(Populus deltoides×Populus nigra) wood. Holzforschung, 2017, 71 (2): 179- 188.
doi: 10.1515/hf-2016-0078 |
|
Yuan H , Zhao L , Guo W , et al. Exogenous application of phytohormones promotes growth and regulates expression of wood formation-related genes in Populus simonii×P. nigra. International Journal of Molecular Sciences, 2019, 20 (3): 792.
doi: 10.3390/ijms20030792 |
|
Zhao C , Craig J C , Petzold H E , et al. The xylem and phloem transcriptomes from secondary tissues of the Arabidopsis root-hypocotyl. Plant Physiology, 2005, 138 (2): 803- 818.
doi: 10.1104/pp.105.060202 |
|
Zheng L , Gao C , Zhao C , et al. Effects of brassinosteroid associated with auxin and gibberellin on apple tree growth and gene expression patterns. Horticultural Plant Journal, 2019, 5 (3): 93- 108.
doi: 10.1016/j.hpj.2019.04.006 |
|
Zhong C , Xu H , Ye S , et al. Gibberellic acid-stimulated Arabidopsis6 serves as an integrator of gibberellin, abscisic acid, and glucose signaling during seed germination in Arabidopsis. Plant Physiology, 2015a, 169 (3): 2288- 2303. | |
Zhong R , Ye Z . Secondary cell walls: biosynthesis, patterned deposition and transcriptional regulation. Plant and Cell Physiology, 2015b, 56 (2SI): 195- 214. | |
Zhou A , Wang H , Walker J C , et al. BRL1, a leucine-rich repeat receptor-like protein kinase, is functionally redundant with BRI1 in regulating Arabidopsis brassinosteroid signaling. The Plant Journal, 2004, 40 (3): 399- 409.
doi: 10.1111/j.1365-313X.2004.02214.x |
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