Research on Mutagenic Mechanism and Genetic Mechanism of a Yellowing Mutant in Barley

doi:10.13304/j.nykjdb.2022.0761

Abstract

Abstract:

In order to study the effect of temperature on phenotype and photosynthetic characteristics of yellowing mutant in barley， the wild-type ‘Yangnongpi 5’ and yellowing mutant G039 obtained by ethyl methanesulfonate （EMS） on two-rowed malting barley ‘Yangnongpi 5’ were used as the test materials in this study. The leaf color， plant main traits， photosynthetic characteristics， chloroplast ultra-structure and photosynthetic related gene expression of mutant and wild type were measured and analyzed under natural field conditions and different indoor temperature. At the same time， the genetic analysis of the mutant gene was carried out by F₁ and F₂ populations that was constructed by crossing the mutant with wild type and ‘Yangnongpi 7’. The results showed that the leaf color of G039 was yellow at seedling stage under natural low temperature， the contents of chlorophyll a and chlorophyll b were significantly lower than ‘Yangnongpi 5’. With the temperature rising， the leaf color gradually turned green. There was no differences in chlorophyll a between G039 and ‘Yangnongpi 5’， but there was still significant differences in chlorophyll b. The plant height， internode length below the spike， number of tillers， panicle length and grains per panicle of G039 were significantly lower than ‘Yangnongpi 5’， while the grain length， grain width and 1 000-grain weight of G039 were significantly higher than ‘Yangnongpi 5’. Under different temperature treatment， G039 was significantly affected by growth temperature. G039 exhibited a less number of chloroplasts and more loose grana lamella at low temperature. Between 10~20 ℃， the colder the temperature was， the more serious the etiolation was， the lower the SPAD value and chlorophyll content were， and the net photosynthetic rate， intercellular CO₂ concentration and water utilization rate of G039 were significantly different from those of ‘Yangnongpi 5’. The optimal temperature for photosynthesis of barley was around 20 °C. Genetic analysis demonstrated that the yellowing trait of G039 was controlled by a single recessive nuclear gene. The qRT-PCR results indicated that genes related to photosynthetic pigment metabolic pathway， chloroplast development and photosystem were significant differences in G039 at different temperatures when compared with those in the wild type. Above results laid the foundation for the analysis mutagenic mechanism and molecular regulation of leaf color mutation in G039.

Key words: barley, mutant, temperature, photosynthetic characteristics

摘要：

为探究温度对大麦黄化突变体表型及光合特性的影响，以二棱啤用大麦品种‘扬农啤5号’及其经甲磺酸乙酯（ethyl methanesulfonate，EMS）诱变筛选到的黄化突变体G039为材料，测定分析田间自然条件和室内不同温度条件下突变体和野生型的叶色、植株主要性状、光合特性、组织结构及光合相关基因的表达；同时利用突变体与野生型及‘扬农啤7号’的杂种F₁和F₂群体，对突变基因进行遗传分析。结果表明，在自然低温条件下，G039苗期叶色呈现黄色，叶绿素a与叶绿素b含量较‘扬农啤5号’极显著降低；随着气温回升，叶色逐渐转绿，G039灌浆期叶绿素a与‘扬农啤5号’无差异，叶绿素b存在显著差异。G039的株高、穗长、穗下节间长、分蘖数与每穗粒数极显著低于‘扬农啤5号’，粒长、粒宽与千粒重极显著高于‘扬农啤5号’。不同温度处理下，突变体G039叶色受生长温度影响显著，低温造成G039叶绿体数目减少，基粒片层疏松；在10~20 ℃之间，温度越低，黄化越严重，SPAD值与叶绿素含量越低，净光合速率、胞间CO₂浓度及水分利用率与‘扬农啤5号’的差异越大，大麦光合作用的最适温度在20 ℃左右。G039表型受1对隐性核基因控制。qRT-PCR结果显示，在不同温度下叶绿素合成、叶绿体发育及光系统建成等相关基因的表达在野生型与突变体G039间均存在极显著差异。以上结果为解析G039叶色突变机理和分子调控机制奠定基础。

关键词: 大麦, 突变体, 温度, 光合特性

CLC Number:

S512.3

Daokuan BAI, Baojian GUO, Yi HONG, Mengna ZHANG, Juan ZHU, Chao LYU, Feifei WANG, Rugen XU. Research on Mutagenic Mechanism and Genetic Mechanism of a Yellowing Mutant in Barley[J]. Journal of Agricultural Science and Technology, 2023, 25(5): 34-45.

白道宽, 郭宝健, 洪益, 张萌娜, 朱娟, 吕超, 王菲菲, 许如根. 一个大麦黄化突变体的突变机理及其遗传机制研究[J]. 中国农业科技导报, 2023, 25(5): 34-45.

Figures/Tables 11

Table 1 Primer of chlorophyll synthesis， photosynthesis related genes used fou quantitative real-time PCR

基因名称 Gene name	基因ID Gene ID	正向引物 Forward primer（5’-3’）	反向引物 Reverse primer（5’-3’）
HvCAO	HORVU.MOREX.r3.3HG0324440	TGGTGCCCATAGATTGTTTC	GCCCTCATTCACTGAACCAAG
COR	HORVU.MOREX.r3.2HG0191600	TCTGCCTGCAAACCCCTCCTAG	TGTCCGTGGCATCCTGGACCTT
PORB	HORVU.MOREX.r3.1HG0047060	AGGCGTACAAGGACAGCAAGG	GTAGAGCGACGCGAAGGTGA
PORA	HORVU.MOREX.r3.2HG0209470	CCGTTCCAGAAGTTCGTCACC	CCTTGTTCCAGCTCCAGTACAC
YCF3	HORVU.MOREX.r3.5HG0423760	GGTCTTTATCCTCGTCGTAT	TGTTCAATTTCTCGTGGAGT
HvPTOX	HORVU.MOREX.r3.2HG0213170	CTCTTAATCATGGAAGCGTTGG	TGGCGACAGTGACGAAGTAG
MSH1	HORVU.MOREX.r3.2HG0180240	CCGAAGAAGAGTTCAGCAAT	TTCCATGCGAGGACATCACG
HvCMF3	HORVU.MOREX.r3.6HG0559110	AATGCGAAATGCGAGGAGG	GGTCTTCGGTGCCTTTGTC
GAPDH	HORVU.MOREX.r3.7HG0703580	GTGAGGCTGGTGCTGATTACG	TGGTGCAGCTAGCATTTGAGA
α-tublin	HORVU.MOREX.r3.1HG0082050	AGTGTCCTGTCCACCCACTC	AGCATGAAGTGGATCCTTGG

Fig. 1 Phenotypes of ‘YNP5’ and G039 under natural conditionA： Plant at seedling period； B： Extracts of photosynthetic pigments in the seedling period； C： Plant at filling period； D： Grain length； E： Grain width

Fig. 2 Contents of chlorophyll pigments in ‘YNP5’ and G039A： Seedling stage； B： Grain filling stage. ** means significantly differences between materials at P<0.01 level

Table 2 Agronomic traits of the wild type ‘YNP5’ and mutant G039

性状 Trait	扬农啤5号 YNP5	G039
株高 Plant height/cm	86.46±5.94	72.86±5.42^**
穗下节间长 Internode length below the spike/cm	30.88±3.36	22.38±2.40^**
分蘖数 Number of tillers	17.82±8.36	8.85±3.40^**
穗长 Panicle length/cm	6.71±0.48	5.95±0.66^**
每穗粒数 Grains per panicle	28.35±2.42	25.86±2.23^**
千粒重 1 000-grain weight/g	41.93±0.34	48.04±0.36^**
粒长 Grain length/mm	6.86±0.03	7.62±0.03^**
粒宽 Grain width/mm	3.05±0.02	3.15±0.02^**

Fig. 3 SPAD values of the ‘YNP5’ and G039 under different temperature conditionsNote：** means significantly difference between materials at P<0.01 level.

Fig. 4 Phenotypes of ‘YNP5’ and G039 under different temperature conditions

Fig. 5 Chlorophyll content of the ‘YNP5’ and G039 under different temperature conditionsNote： ** means significantly difference between materials at P<0.01 level

Fig. 6 Photosynthetic indices of ‘YNP 5’ and G039 under different temperature conditionsA~E： Net photosynthetic rate， transpiration rate， stomatal conductance， water use efficiency and intercellular CO2 concentration are sequentially shown. * and ** mean significantly differences between materials at P<0.05 and P<0.01 levels，respectively

Fig. 7 Ultrastructure of chloroplasts in cells of G039 mutant under different temperatures conditions

Table 3 Genetic analysis of the mutant trait

杂交组合

Cross combination

植株数量 Number of plants

χ²

P值（0.05）

P value（0.05）

F₂群体

F₂ population

野生表型

Wild type phenotype

突变表型

Mutant phenotype

Fig. 8 Expression levels in genes involved in chlorophyll synthesis of YNP 5 and G039 under different temperature conditionsNote： ** means significant at P<0.01 level.

References 33

1	李强强,赵玥,李璠,等.作物源库关系及其生理调控途径的研究进展[J].江苏农业科学,2020,48(9):50-56.
	LI Q Q, ZHAO Y, LI F, et al.. Research progress of source-sink relationship and its underlying regulation mechanisms [J]. Jiangsu Agric. Sci., 2020, 48(9): 50-56.
2	GAO F, ZHANG H J, ZHANG W J, et al.. Engineering of the cytosolic form of phosphoglucose isomerase into chloroplasts improves plant photosynthesis and biomass [J]. New Phytol., 2021, 231(1) : 315-325.
3	李佳佳,于旭东,蔡泽坪,等.高等植物叶绿素生物合成研究进展[J].分子植物育种,2019,17(18):6013-6019.
	LI J J, YU X D, CAI Z P, et al.. An overview of chlorophyll biosynthesis in higher plants [J]. Mol. Plant Breed., 2019, 17(18): 6013-6019.
4	李素贞,杨文竹,陈茹梅.水稻黄绿叶突变体研究进展[J].生物技术通报,2018,34(11):15-21.
	LI S Z, YANG W Z, CHEN R M. An overview on yellow green leaf mutants in rice [J]. Biotechnol. Bull., 2018, 34(11): 15-21.
5	李军,朱梦珂,田激旋,等.水稻高温白化复绿突变体tcd52的遗传分析和分子定位[J].上海师范大学学报(自然科学版),2022,51(2):243-250.
	LI J, ZHU M K, TIAN J X, et al.. Genetic analysis and molecular mapping of high-temperature albino regreen mutant tcd52 in rice [J]. J. Shanghai Normal Univ. (Nat. Sci.), 2022, 51(2): 243-250.
6	莫祎,孙志忠,丁佳,等.水稻白条纹叶突变体wsl1的遗传分析及基因精细定位[J].作物学报,2019,45(7):1050-1058.
	MO Y, SUN Z Z, DING J, et al.. Genetic analysis and fine mapping of white stripe leaf mutant wsl1 in rice [J]. Acta Agron. Sin., 2019, 45(7): 1050-1058.
7	鄢小青,陈能刚,李欢,等.水稻白条纹转绿突变体wsl887的鉴定和基因定位[J].核农学报, 2021, 35(11) : 2451-2462.
	YAN X Q, CHEN N G, LI H, et al.. Phenotypic identification and gene mapping of green revertible white-stripes mutant wsl887 in rice (Oryza sativa L.) [J]. J. Nucl. Agric. Sci., 2021, 35(11): 2451-2462.
8	郭均瑶,刘斌美,杨惠杰,等.水稻叶脉黄化突变体yml的遗传分析及基因定位[J].作物学报, 2022, 48(12): 3120-3129.
	GUO J Y, LIU B M, YANG H J, et al.. Genetic analysis and gene mapping of the yellow midrib leaf mutant (yml) in rice(Oryza sativa L.) [J]. Acta Agron. Sin., 2022, 48(12): 3120-3129.
9	李秦,杜何为.玉米叶色突变体研究进展[J].南方农业,2019,13(28):14-21, 7.
	LI Q, DU H W. Advances in research on maize leaf color mutants [J]. South Chin. Agric., 2019, 13(28): 14-21, 7.
10	REINBOTHE S, REINBOTHE C, HOLTORF H, et al.. Two NADPH: protochlorophyllide oxidoreductases in barley: evidence for the selective disappearance of PORA during the light-induced greening of etiolated seedlings [J]. Plant Cell, 1995,7(11):1933-1940.
11	BUHR F, LAHROUSSI A, SPRINGER A, et al.. NADPH: protochlorophyllide oxidoreductase B (PORB) action in Arabidopsis thaliana revisited through transgenic expression of engineered barley PORB mutant proteins [J]. Plant Mol. Biol., 2017, 94(1-2):45-59.
12	XU D D, SUN D, DIAO Y L, et al.. Fast mapping of a chlorophyll b synthesis-deficiency gene in barley (Hordeum vulgare L.) via bulked-segregant analysis with reduced-representation sequencing [J]. Crop J., 2019, 7(1): 58-64.
13	MAHDI R, STUART D, HANSSON M, et al.. Heterologous expression of the barley (Hordeum vulgare L.) Xantha-f, -g and -h genes that encode magnesium chelatase subunits [J]. Protein J., 2020, 39(5):554-562.
14	LI M J, HENSEL G, MASCHER M, et al.. Leaf variegation and impaired chloroplast development caused by a truncated CCT domain gene in albostrians barley [J]. Plant Cell, 2019, 31(7) : 1430-1445.
15	LI M J, HENSEL G, MELZER M, et al.. Mutation of the ALBOSTRIANS ohnologous gene HvCMF3 impairs chloroplast development and thylakoid architecture in barley [J]. Front. Plant Sci., 2021, 12: 732608-732630.
16	LI M J, GUO G G, PIDON H, et al.. ATP-dependent Clp protease subunit C1, HvClpC1, is a strong candidate gene for barley variegation mutant luteostrians as revealed by genetic mapping and genomic re-sequencing [J/OL]. Front. Plant Sci., 2021,12:664085 [2022-11-16]. .
17	LANDAU A M, LOKSTEIN H, SCHELLER H V, et al.. A cytoplasmically inherited barley mutant is defective in photosystem Ⅰ assembly due to a temperature-sensitive defect in ycf3 splicing [J]. Plant Physiol., 2009, 151(4): 1802-1811.
18	LENCINA F, LANDAU A, PRINA A R. The barley chloroplast mutator (cpm) mutant: all roads lead to the Msh1 gene [J]. Int. J. Mol. Sci., 2022, 23(3):1814-1828.
19	BOSCO C D, BUSCONI M, GOVONI C, et al.. cor gene expression in barley mutants affected in chloroplast development and photosynthetic electron transport [J]. Plant Physiol., 2003,131(2):793-802.
20	QIN D D, DONG J, XU F C, et al.. Characterization and fine mapping of a novel barley stage green-revertible albino gene (HvSGRA) by bulked segregant analysis based on SSR assay and specific length amplified fragment sequencing [J]. BMC Genomics, 2015, 16: 838-851.
21	ROTASPERTI L, SANSONI F, MIZZOTTI C, et al.. Barley’s second spring as a model organism for chloroplast research [J]. Plants (Basel), 2020, 9(7): 803-826.
22	WANG R, YANG F, ZHANG X Q, et al.. Characterization of a thermo-inducible chlorophyll-deficient mutant in barley [J]. Front. Plant Sci., 2017, 8: 1936-1944.
23	闫伟平,赵洪祥,张丽华,等.半干旱区温度变化对不同密度玉米植株光合作用的影响[J].吉林农业科学,2015,40(5):14-20.
	YAN W P, ZHAO H X, ZHANG L H, et al.. Effect of temperature changes rates of maize under different density in semiarid area [J]. Jilin Agric. Sci., 2015, 40(5): 14-20.
24	江华,师生波,许大全.冬季小麦叶片光合作用对温度响应方式的变化[J].植物生理学报,2000,26(1):70-75.
	JIANG H, SHI S B, XU D Q. Change in the pattern of photosynthetic response to temperature in wheat leaves in winter [J]. Plant Physiol. J., 2000, 26(1): 70-75.
25	杨淑巧,许琦,刘跃鹏,等.冬小麦光合作用和叶绿素荧光特性的研究[J].农学学报,2015,5(3):5-10.
	YANG S Q, XU Q, LIU Y P, et al.. Research on the photosynthesis and chlorophyll fluorspar characteristic of winter wheat [J]. J. Agric., 2015, 5(3): 5-10.
26	闫蓉,李凤霞,赵维忠,等.气象条件对水稻蒸腾速率的影响[J].宁夏农林科技,2005(2):7-8, 10.
	YAN R, LI F X, ZHAO W Z, et al.. Effect of meteorological conditions on transpiration rate of rice (Oryza sativa L.) [J]. Ningxia J. Agric. For., 2005 (2): 7-8, 10.
27	董树亭,胡昌浩,周关印.玉米叶片气孔导度、蒸腾和光合特性研究[J].玉米科学,1993,1(2):41-44.
	DONG S T, HU C H, ZHOU G Y. Stomatal conductance, transpiration and photosynthetic characteristics of maize leaves [J]. J. Maize Sci., 1993, 1(2): 41-44.
28	刘亮,郝立华,李菲,等. CO₂浓度和温度对玉米光合性能及水分利用效率的影响[J].农业工程学报,2020,36(5):122-129.
	LIU L, HAO L H, LI F, et al.. Effects of CO₂ concentration and temperature on leaf photosynthesis and water use efficiency in maize [J]. Trans. Chin. Soc. Agric. Eng., 2020, 36(5): 122-129.
29	KUSUMI K, HIROTSUKA S, SHIMADA H, et al.. Contribution of chloroplast biogenesis to carbon-nitrogen balance during early leaf development in rice [J]. Plant Res., 2010, 123 (4): 617-622.
30	SUGIMOTO H, KUSUMI K, NOGUCHI K, et al.. The rice nuclear gene, VIRESCENT 2, is essential for chloroplast development and encodes a novel type of guanylate kinase targeted to plastids and mitochondria [J]. Plant J., 2007, 52 (3): 512-527.
31	HUANG W F, ZHANG Y, SHEN L Q, et al.. Accumulation of the RNA polymerase subunit RpoB depends on RNA editing by OsPPR16 and affects chloroplast development during early leaf development in rice [J]. New Phytol., 2020, 228(4): 1401-1416.
32	HEIN P, STÖCKEL J, BENNEWITZ S, et al.. A protein related to prokaryotic UMP kinases is involved in psaA/B transcript accumulation in Arabidopsis [J]. Plant Mol. Biol., 2009, 69 (5) : 517-528.
33	YANG S M, OVERLANDER M, FIEDLER J. Genetic analysis of the barley variegation mutant, grandpa1.a [J]. BMC Plant Biol., 2021, 21(1) : 134-144.

[1]	Haitao XU, Hongzhen MA, Wenwen WANG, Wenxiang FAN, Bo XU, Jungang ZHANG, Haibin GUO, Youhua WANG. Research on Dynamic Development and Accumulated Temperature Model of Maize Plant Height and Stem Diameter Based on Effective Accumulated Temperature [J]. Journal of Agricultural Science and Technology, 2025, 27(8): 187-201.
[2]	Yanfang ZHU, Qiang CHANG, Yan HAO, Hailong CHEN. Effects of Reflective Film on Fruit Quality and Volatile Components of ‘Shine Muscat’ Grape [J]. Journal of Agricultural Science and Technology, 2025, 27(7): 72-82.
[3]	Taotao MAO, Xiaoqiang ZHAO, Xiaodong BAI, Bin YU. Effect of Low Temperature Stress on Photosynthetic Performance， Antioxidant Enzyme System and Related Gene Expression in Maize Seedlings [J]. Journal of Agricultural Science and Technology, 2025, 27(5): 49-60.
[4]	Xiaoqing HOU, Zihao JIANG, Yang FU, Zhongzhen SONG, Zhimin YU. Composition and Structure of Extracellular Polysaccharides from Sphingomonas sp. and Their Promoting Effects on Barley Seedlings [J]. Journal of Agricultural Science and Technology, 2025, 27(4): 201-208.
[5]	Bei MA, Jie GONG, Yinke DU, Yuwei GAN, Rong CHENG, Bo ZHU, Lixia YI, Jinxiu MA, Shiqing GAO. Identification and Expression Analysis of TaINP1 Gene Related to Pollen Pore Development in Wheat [J]. Journal of Agricultural Science and Technology, 2025, 27(4): 22-35.
[6]	Xiaodan WU, Li GAO, Tiangeng GONG, Xiangfeng KONG, Yuzhou JIANG, Guixia JIA. Effects of Microbial Fertilizer and Humic Acid Compound Fertilizer on Growth and Photosynthetic Characteristics of Lilium [J]. Journal of Agricultural Science and Technology, 2025, 27(4): 221-229.
[7]	Yanxia MA, Jingru CHEN, Xiaowei WANG, Yuxin ZHANG, Junfeng ZHANG, Jialin KUAI. Effects of Irrigation Lower Limit and Fertilizer Application Amount on Water Consumption and Photosynthetic Characteristics of Mini Chinese Cabbage Under Drip Irrigation [J]. Journal of Agricultural Science and Technology, 2025, 27(3): 239-249.
[8]	Zhiwei LYU, Dongmei LI, Meijuan JIN, Yanhui ZHANG, Yueyue TAO, Xinwei ZHOU, Haihou WANG. Effects of Pyrolysis Temperature and Time on Physicochemical Properties and Adsorption Properties of Biochar [J]. Journal of Agricultural Science and Technology, 2025, 27(2): 211-217.
[9]	Lanting XIANG, Shuhui SONG, Lijuan LIU, Xiaoling SHE, Jiahua ZHOU, Baogang WANG, Hong CHANG, Chao ZHANG, Daqi FU, Yunxiang WANG. Effect of Different Storage Temperatures on Quality of Jingcai 1 Watermelon [J]. Journal of Agricultural Science and Technology, 2024, 26(9): 137-145.
[10]	Xiaoyu SHI, Lianqing JIAO, Min YU, Yixin TIAN, Anni JIAO, Yilin LUAN. Multidimensional Evaluation and Optimization of High Temperature Short Time Process of Astragalus membranaceus [J]. Journal of Agricultural Science and Technology, 2024, 26(7): 223-233.
[11]	Xiaofei XIONG, Wenqian WU, Hongyan HUO, Xin ZHANG, Yan YU, Dong AN, Tong ZHANG, Jianwei WU. Research on Sensor-based Agricultural Greenhouse Data Direct Reporting System and Intelligent Control [J]. Journal of Agricultural Science and Technology, 2024, 26(7): 93-102.
[12]	Zhongxiang LIU, Wenqi ZHOU, Yongsheng LI, Xiaojuan WANG, Yanzhong YANG, Xiaorong LIAN, Haijun HE, Yuqian ZHOU. Phenotypic Identification and Genetic Analysis of a Dwarf Mutant 20F421 inMaize [J]. Journal of Agricultural Science and Technology, 2024, 26(6): 22-29.
[13]	Suilin ZHANG, Yang LI, Yan LI, Yunqi ZHANG, Jianxun QI, Zhixia HOU. Research Progress on Harmful Characteristics and Mechanism of Walnut Late Frost [J]. Journal of Agricultural Science and Technology, 2024, 26(4): 18-26.
[14]	Jianguang ZENG, Taoli LIU, Linjuan SUN, Dingyang YUAN, Yubo HUANG, Chenzhong JIN, Yanning TAN. Analysis of Character and GibberellinSensitivity of Rice Dwarfism and Late-Heading Mutant d534 [J]. Journal of Agricultural Science and Technology, 2024, 26(3): 7-14.
[15]	Zhongyi LI, Hongqin TANG, Wenbin DONG, Caihui WEI, Tieguang HE. Effects of Co-incorporation of Rice Straw and Chinese Milk Vetch on Photosynthetic Characteristics， Yield and Quality of Rice [J]. Journal of Agricultural Science and Technology, 2024, 26(2): 171-180.