高粱表皮蜡质缺失突变体sb1抗旱生理生化分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0889

摘要/Abstract

摘要：

植物抗旱性与体表蜡质积累有关，高粱是抗旱性极强的作物，茎秆和叶片表面覆盖一层厚厚的蜡质，开展高粱体表蜡质层对高粱抗旱能力的研究，为高粱抗旱品种选育和抗旱分子机制提供理论依据。以高粱品种BTx623和表皮蜡质缺失突变体sb1为试验材料，统计农艺性状以及扫描电镜观察叶片表面蜡质形态结构；分析表皮蜡质的有无对离体叶片失水速率和叶绿素浸提率的影响；检测在干旱胁迫处理下植株的表型变化以及超氧化物歧化酶（SOD）、过氧化物酶（POD）的活性变化。结果表明，与对照BTx623相比，表皮蜡质缺失突变体sb1株高明显降低、抽穗开花期延后1周、叶片远轴面片状蜡质缺失，其他农艺性状变化不明显。突变体sb1离体叶片失水速率和叶绿素浸提率显著高于对照。在干旱胁迫处理条件下，随着干旱胁迫时间的增长，对照BTx623和突变体sb1的SOD和POD活性均增强，但对照SOD和POD活性均显著高于突变体sb1，且干旱处理96 h和复水后突变体sb1都造成叶片大面积干枯。突变体sb1表皮蜡质缺失后叶片渗透性增强，清除活性氧的能力减弱，抗旱性降低。

关键词: 高粱, 表皮蜡质, 干旱胁迫, 渗透性, 抗氧化酶

Abstract:

The drought tolerance trait of plant is related to the wax accumulation on the surface. Sorghum is a cereal crop that can survive under extreme drought stress, its surfaces of stems and leaves were covered with a thick layer of wax on the. The study on the difference of drought resistance in sorghum with or without cuticular wax may provide a theoretical basis for breeding drought-resistant sorghum varieties and revealing the molecular mechanism of drought resistance. Sorghum cv. BTx623（with cuticular wax）and cuticular wax-deficient mutant sb1（without cuticular wax）were used as experimental materials. Firstly, agronomic traits of BTx623 and sb1 were recoded and leaf surface wax structure was observed by using electron microscope. Secondly, the influences of the presence and absence of cuticular wax on the water loss rates of excised leaves and chlorophyll leaching rates were analyzed. Finally, phenotype changes, activities of SOD（superoxide dismutase）and POD（peroxidase）were detected under drought stress. Compared to sorghum cv. BTx623, the height of cuticular wax-deficient mutant sb1 significantly decreased, while the heading-flowering period delayed for one week. Sheticular wax was absent on the distal axial surface of leaves, and no significant variance in other agronomic traits was found. Water loss rates of excised leaves and chlorophyll leaching rates of mutant sb1 were significantly higher than those of BTx623. Under the drought stress treatment, the activities of SOD and POD were induced in both BTx623 and sb1, while the enzyme activities of BTx623 were significantly higher than those of sb1. After drought treatment, most leaves of sb1 were withered and dried after 96 h with re-watering. This study demonstrate that leaf's permeability is enhanced and leaf's ability to scavenge reactive oxygen species is weakened in cuticular wax-deficient mutant sb1 compared to BTx623, which indicates the presence of cuticular wax plays an important role in drought tolerance.

Key words: sorghum, cuticular wax, drought stress, permeability, antioxidant enzyme

王春语, 李政君, 王平, 张丽霞. 高粱表皮蜡质缺失突变体sb1抗旱生理生化分析[J]. 生物技术通报, 2023, 39(5): 160-167.

WANG Chun-yu, LI Zheng-jun, WANG Ping, ZHANG Li-xia. Physiological and Biochemical Analysis of Drought Resistance in Sorghum Cuticular Wax-deficient Mutant sb1[J]. Biotechnology Bulletin, 2023, 39(5): 160-167.

图/表 6

图1 抽穗开花期BTx623与sb1的表型 A：整株；B：叶片近轴面；C：叶片远轴面

Fig. 1 Phenotypes of BTx623 and sb1 in heading and flow-ering period A: The whole plants. B: The adaxial sides of leaves. C: The abaxial sides of leaves

图2 BTx623和sb1叶片远轴面扫描电镜观察

Fig. 2 Observation of the abaxial sides of leaves between BTx623 and sb1 by scanning electron microscope

表1 BTx623和sb1农艺性状比较

Table 1 Comparison of agronomic traits between BTx623 and sb1

材料名称 Genotype	株高 Plant height/cm	茎粗 Stem diameter/cm	穗长 Spike length/cm	穗茎长 Spike stem length/cm	粒数 Grain yield per plant	千粒重 1 000-grain weight/g	抽穗期 Heading stage/d
BTx623	138.67±5.51	1.63±0.08	32.33±2.52	37.67±2.08	1 612.52±39.23	26.19±0.21	79±2.16
sb1	133.97±6.10*	1.62±0.14	33.32±0.89	36.33±1.52	1 487.08±28.59	25.06±0.43	86±2.46*

图3 BTx623和sb1叶片渗透性分析 A：离体叶片失水速率；B：离体叶片失水表型；C：叶绿素浸提率。**表示在0.01水平上差异显著。下同

Fig. 3 Leaf permeabilities between BTx623 and sb1 A: Water loss rates of excised leaves. B: Phenotypes of excised-leaves water loss. C: Chlorophyll leaching rates. ** indicates significant difference at the 0.01 level. The same as below

图4 干旱胁迫处理下BTx623和sb1的表型变化 A：正常浇水；B：干旱胁迫处理72 h；C：干旱胁迫处理96 h；D：复水后96 h

Fig. 4 Phenotypes of BTx623 and sb1 under drought stress A: Normal watering. B: Drought stress for 72 h. C: Drought stress for 96 h.D: Seedlings recovery 96 h after re-watering

图5 干旱胁迫处理后BTx623和sb1抗氧化酶活性

Fig. 5 Antioxidant enzyme activities of BTx623 and sb1 under drought stress

参考文献 31

[1]	Menezes CB, Ticona-Benavente CA, Tardin FD, et al. Selection indices to identify drought-tolerant grain sorghum cultivars[J]. Genet Mol Res, 2014, 13(4): 9817-9827. doi: 10.4238/2014.November.27.9 pmid: 25501191
[2]	Dahlberg J. The role of sorghum in renewables and biofuels[J]. Methods Mol Biol, 2019, 1931: 269-277. doi: 10.1007/978-1-4939-9039-9_19 pmid: 30652297
[3]	Gladman N, Hufnagel B, Regulski M, et al. Sorghum root epigenetic landscape during limiting phosphorus conditions[J]. Plant Direct, 2022, 6(5): e393. doi: 10.1002/pld3.393 pmid: 35600998
[4]	张福耀, 赵威军, 平俊爱. 高能作物——甜高粱[J]. 中国农业科技导报, 2006, 8(1): 14-17.
	Zhang FY, Zhao WJ, Ping JN. Macroergic crop—sweet sorghum[J]. Rev China Agric Sci Technol, 2006, 8(1): 14-17.
[5]	裴冬, 张喜英, 王峻. 高粱、谷子根系发育及其抗旱性研究[J]. 中国生态农业学报, 2002, 10(4): 28-30.
	Pei D, Zhang XY, Wang J. Study on root system development and drought resistance of sorghum and millet[J]. Chin J Eco Agric, 2002, 10(4): 28-30.
[6]	Elango D, Xue WY, Chopra S. Genome wide association mapping of epi-cuticular wax genes in Sorghum bicolor[J]. Physiol Mol Biol Plants, 2020, 26(8): 1727-1737. doi: 10.1007/s12298-020-00848-5
[7]	Xue DW, Zhang XQ, Lu XL, et al. Molecular and evolutionary mechanisms of cuticular wax for plant drought tolerance[J]. Front Plant Sci, 2017, 8: 621. doi: 10.3389/fpls.2017.00621 pmid: 28503179
[8]	Jenks MA, Eigenbrode SD, Lemieux B. Cuticular waxes of Arabidopsis[J]. Arabidopsis Book, 2002, 1: e0016. doi: 10.1199/tab.0016 URL
[9]	Zhao Y, Liu XJ, Wang MK, et al. Transcriptome and physiological analyses provide insights into the leaf epicuticular wax accumulation mechanism in yellowhorn[J]. Hortic Res, 2021, 8(1): 134. doi: 10.1038/s41438-021-00564-5
[10]	韦百阳, 徐小静. 植物表皮蜡质参与干旱胁迫的反应机制[J]. 生物技术通报, 2015, 31(8): 1-8. doi: 10.13560/j.cnki.biotech.bull.1985.2015.08.001
	Wei BY, Xu XJ. Response mechanism of plant cuticular wax involving in drought stress response[J]. Biotechnol Bull, 2015, 31(8): 1-8. doi: 10.13560/j.cnki.biotech.bull.1985.2015.08.001
[11]	Lewandowska M, Keyl A, Feussner I. Wax biosynthesis in response to danger: its regulation upon abiotic and biotic stress[J]. New Phytol, 2020, 227(3): 698-713. doi: 10.1111/nph.16571 pmid: 32242934
[12]	Tomasi P, Wang H, Lohrey GT, et al. Characterization of leaf cuticular waxes and cutin monomers of Camelina sativa and closely-related Camelina species[J]. Ind Crops Prod, 2017, 98: 130-138. doi: 10.1016/j.indcrop.2017.01.030 URL
[13]	Zhang XF, Ni Y, Xu DX, et al. Integrative analysis of the cuticular lipidome and transcriptome of Sorghum bicolor reveals cultivar differences in drought tolerance[J]. Plant Physiol Biochem, 2021, 163: 285-295. doi: 10.1016/j.plaphy.2021.04.007 URL
[14]	Vogg G, Fischer S, Leide J, et al. Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid beta-ketoacyl-CoA synthase[J]. J Exp Bot, 2004, 55(401): 1401-1410. doi: 10.1093/jxb/erh149 pmid: 15133057
[15]	Seo PJ, Lee SB, Suh MC, et al. The MYB96 transcription factor regulates cuticular wax biosynthesis under drought conditions in Arabidopsis[J]. Plant Cell, 2011, 23(3): 1138-1152. doi: 10.1105/tpc.111.083485 URL
[16]	Zhang Y, Du ZH, Han YT, et al. Plasticity of the cuticular transpiration barrier in response to water shortage and resupply in Camellia sinensis: a role of cuticular waxes[J]. Front Plant Sci, 2021, 11: 600069. doi: 10.3389/fpls.2020.600069 URL
[17]	张海禄, 齐军仓, 王祥军. 干旱胁迫对大麦叶片表皮蜡质含量及主要生理指标的影响[J]. 麦类作物学报, 2012, 32(2): 280-283.
	Zhang HL, Qi JC, Wang XJ. Effects of water stress on epicuticular wax content and main physiological parameters of barley[J]. J Triticeae Crops, 2012, 32(2): 280-283.
[18]	徐文. 旗叶蜡质含量不同小麦近等基因系的抗旱性研究[D]. 泰安: 山东农业大学, 2016.
	Xu W. Drought resistance of wheat NILs with different cuticular wax content in flag leaf[D]. Tai'an: Shandong Agricultural University, 2016.
[19]	甘露. 水稻蜡质合成代谢基因WSL3及WSL4的克隆和功能分析[D]. 北京: 中国农业科学院, 2016.
	Gan L. Map-based cloning and functional analysis of WSL3 and WSL4 involved in cuticular wax biosythesis in rice[D]. Beijing: Chinese Academy of Agricultural Sciences, 2016.
[20]	毛毕刚. 水稻叶片蜡质基因的图们克隆与功能分析[D]. 北京: 中国农业科学院, 2010.
	Mao BG. Map-based cloning and functional analysis of leaf wax gene in rice(Orzya sativa L.)[D]. Beijing: Chinese Academy of Agricultural Sciences, 2010.
[21]	Sanjari S, Shobbar ZS, Ghanati F, et al. Molecular, chemical, and physiological analyses of sorghum leaf wax under post-flowering drought stress[J]. Plant Physiol Biochem, 2021, 159: 383-391. doi: 10.1016/j.plaphy.2021.01.001 URL
[22]	Zhang ZZ, Wang W, Li WL. Genetic interactions underlying the biosynthesis and inhibition of β-diketones in wheat and their impact on glaucousness and cuticle permeability[J]. PLoS One, 2013, 8(1): e54129. doi: 10.1371/journal.pone.0054129 URL
[23]	Lolle SJ, Berlyn GP, Engstrom EM, et al. Developmental regulation of cell interactions in the Arabidopsis fiddlehead-1 mutant: a role for the epidermal cell wall and cuticle[J]. Dev Biol, 1997, 189(2): 311-321. doi: 10.1006/dbio.1997.8671 pmid: 9299123
[24]	潘孝武, 黎用朝, 刘文强, 等. 水稻蜡质稀少突变体wax1的鉴定及基因定位[J]. 中国水稻科学, 2020, 34(1): 1-7. doi: 10.16819/j.1001-7216.2019.9101
	Pan XW, Li YC, Liu WQ, et al. Identification and genetic analysis of waxy sparse mutant wax1 in rice[J]. Chin J Rice Sci, 2020, 34(1): 1-7.
[25]	Mwamahonje A, Eleblu JSY, Ofori K, et al. Drought tolerance and application of marker-assisted selection in sorghum[J]. Biology, 2021, 10(12): 1249. doi: 10.3390/biology10121249 URL
[26]	Busta L, Schmitz E, Kosma DK, et al. A co-opted steroid synthesis gene, maintained in sorghum but not maize, is associated with a divergence in leaf wax chemistry[J]. Proc Natl Acad Sci USA, 2021, 118(12): e2022982118. doi: 10.1073/pnas.2022982118 URL
[27]	赖勇. 大麦EMS诱变蜡质突变体鉴定分析[D]. 兰州: 甘肃农业大学, 2014.
	Lai Y. Characeration and analysis of wax mutant induced by EMS in barley[D]. Lanzhou: Gansu Agricultural University, 2014.
[28]	党云萍, 李春霞, 刘东雄. 水分胁迫对植物生理生化研究进展[J]. 陕西农业科学, 2012, 58(5): 89-93, 122.
	Dang YP, Li CX, Liu DX. Research progress of water stress on plant physiology and biochemistry[J]. Shaanxi J Agric Sci, 2012, 58(5): 89-93, 122.
[29]	Fiebig A, Mayfield JA, Miley NL, et al. Alterations in CER6, a gene identical to CUT1, differentially affect long-chain lipid content on the surface of pollen and stems[J]. Plant Cell, 2000, 12(10): 2001-2008. doi: 10.1105/tpc.12.10.2001 pmid: 11041893
[30]	郑好, 吕夏晨, 谭赛琼, 等. 干旱胁迫下大麦蜡质缺失突变体的生理生化指标及蜡质基因表达[J]. 浙江大学学报: 农业与生命科学版, 2019, 45(1): 8-13.
	Zheng H, Lü XC, Tan SQ, et al. Physiological and biochemical indexes and waxy gene expression of wax-deficient mutant in barley under drought stress[J]. J Zhejiang Univ Agric Life Sci, 2019, 45(1): 8-13.
[31]	裴冬丽, 张红岩, 张贺, 等. 干旱胁迫对番茄幼苗叶片SOD、POD和PAL活性的影响[J]. 吉林农业科学, 2015, 40(4): 83-86.
	Pei DL, Zhang HY, Zhang H, et al. Effects of drought stress on SOD, POD and PAL activity in tomato seedling leaves[J]. J Jilin Agric Sci, 2015, 40(4): 83-86.