Phenotype and biomass analysis of nodulation-deletion mutants in Melilotus albus

doi:10.11686/cyxb2022464

Abstract

Abstract:

In order to elucidate the mechanism of symbiotic nitrogen fixation of Melilotus， nodulation-deletion mutants Ma58， Ma61 and Ma62 induced by ethylmethane sulfonate （EMS）， and wild-type Ma389 of Melilotus albus were studied and the nodulation phenotypes and biomass were analyzed after inoculation with Sinorhizobium meliloti SM1021. It was found that mutants Ma58 and Ma61 did not form infection lines and nodule primordia， while mutant Ma62 did not form infection lines and only formed a few small white nodules which did not fix nitrogen. The root fresh/dry weight of the three mutants was significantly lower than that of the wild type in low nitrogen substrate for 30 d （P<0.05）， and the aboveground fresh/dry weight， root fresh/dry weight， plant height and root length were significantly lower than those of the wild type from 40 d （P<0.05）. However， no significant differences were found when plants were grown in a nitrogen-rich substrate， indicating that the mutant genes of the three mutants were related only to symbiotic nitrogen fixation. This study has identified genetic resources for the study of the symbiotic nitrogen fixation mechanisms of Melilotus.

Key words: Melilotus albus, nodulation-deletion mutants, symbiotic nitrogen fixation, biomass

Sheng-sheng WANG, Zhen DUAN, Pei ZHOU, Ji-yu ZHANG. Phenotype and biomass analysis of nodulation-deletion mutants in Melilotus albus[J]. Acta Prataculturae Sinica, 2023, 32(10): 247-256.

Figures/Tables 8

Fig.1 Responses of root hairs from wild-type and three mutants inoculated with SM1021

Fig. 2 Root nodulation phenotypes of wild-type and three mutants at 30 days

Fig.3 Nodule number of wild-type and three mutants at 30 days

Fig.4 Root nodule section phenotype of wild-type Ma389 and mutant Ma62

Fig.5 Biomass of wild-type and three mutants in low nitrogen substrate

Fig.6 Phenotypes of wild-type and three mutants at 30 days in low nitrogen substrate

Fig.7 Biomass of wild-type and three mutants in rich nitrogen substrate

Fig.8 Phenotypes of wild-type and three mutants at 30 days in rich nitrogen substrate

References 37

1	Wang J Y. Screening and functional verification of genes related to symbiotic nitrogen fixation in mesorhizobium MAFF303099. Wuhan： Huazhong Agricultural University， 2021.
	王婧仪. 中慢生根瘤菌MAFF303099共生固氮相关基因筛选及功能验证. 武汉：华中农业大学， 2021.
2	Zheng H L， Pang B P， Jin R S， et al. Primary identification of nitrogen fixation bacteria isolated from rhizosphere of 4 species of grasses in Xilingol grassland of Inner Mongolia. Journal of Arid Land Resources and Environment， 2011， 25（9）： 106-109.
	郑红丽，庞保平，靳润岁，等. 内蒙古锡林郭勒天然草原禾本科牧草根际18株固氮细菌的初步分类鉴定. 干旱区资源与环境， 2011， 25（9）： 106-109.
3	Huo H B. Responses of Robinia pseudoacacia to rhizobial nod factor and the mechanism of heme degrading enzyme HmuS in symbiosis. Xianyang： Northwest A & F University， 2021.
	霍海波. 刺槐对根瘤菌结瘤因子的响应及血红素降解因子HmuS在共生中的作用机制研究. 咸阳：西北农林科技大学， 2021.
4	Zhang C. The phenotypic analysis and map-based cloning of nitrogen fixing deficient mutants in Medicago truncatula. Changsha： Hunan University， 2021.
	张超. 蒺藜苜蓿固氮缺失突变体的表型分析和图位克隆. 长沙：湖南大学， 2021.
5	Cui Z L， Zhang H Y， Chen X P， et al. Pursuing sustainable productivity with millions of smallholder farmers. Nature， 2018， 555（7696）： 363-380.
6	Zhang X， Eric A D， Denise L M， et al. Managing nitrogen for sustainable development. Nature， 2015， 528（5743）： 51-59.
7	Xie K Y， Wang Y X， Wan J C， et al. Mechanisms and factors affecting nitrogen transfer in mixed legume/grass swards： A review. Acta Prataculturae Sinica， 2020， 29（3）： 157-170.
	谢开云，王玉祥，万江春，等. 混播草地中豆科/禾本科牧草氮转移机理及其影响因素. 草业学报， 2020， 29（3）： 157-170.
8	Doyle J J， Luckow M A. The rest of the iceberg. Legume diversity and evolution in a phylogenetic context. Plant Physiology， 2003， 131（3）： 900-910.
9	Li J H， Xi N， Man J， et al. Effects rhizobial population and inoculation method on the efficiency of alfalfa rhizobium inoculation. Acta Agrestia Sinica， 2022， 30（3）： 743-749.
	李佳欢，希娜，漫静，等. 苜蓿根瘤菌接种数量与方式对接种效果的影响. 草地学报， 2022， 30（3）： 743-749.
10	Sutton M A， Oenema O， Erisman J W， et al. Too much of a good thing. Nature， 2011， 472（7342）： 159-161.
11	Peoples M B， Brockwell J， Herridge D F， et al. The contributions of nitrogen-fixing crop legumes to the productivity of agricultural systems. Symbiosis， 2009， 48（1/2/3）： 1-17.
12	Roy S， Liu W， Nandety R S， et al. Celebrating 20 years of genetic discoveries in legume nodulation and symbiotic nitrogen fixation. Plant Cell， 2020， 32（1）： 15-41.
13	Saito K， Yoshikawa M， Yano K， et al. NUCLEOPORIN85 is required for calcium spiking， fungal and bacterial symbioses， and seed production in Lotus japonicus. Plant Cell， 2007， 19（2）： 610-624.
14	Kiss E， Olah B， Kalo P， et al. LIN， a novel type of U-Box/WD40 protein， controls early infection by rhizobia in legumes. Plant Physiology， 2009， 151（3）： 1239-1249.
15	Peters J L， Cnudde F， Gerats T. Forward genetics and map-based cloning approaches. Trends in Plant Science， 2003， 8（10）： 484-491.
16	Stracke S， Kistner C， Yoshida S， et al. A plant receptor-like kinase required for both bacterial and fungal symbiosis. Nature， 2002， 417（6892）： 959-962.
17	Wang D， Griffitts J， Starker C， et al. A nodule-specific protein secretory pathway required for nitrogen-fixing symbiosis. Science， 2010， 327（5969）： 1126-1129.
18	Catoira R， Galera C， de Billy F， et al. Four genes of Medicago truncatula controlling components of a nod factor transduction pathway. Plant Cell， 2000， 12（9）： 1647-1665.
19	Di H Y， Luo K， Zhang J Y， et al. Genetic diversity analysis of Melilotus populations based on ITS and trnL-trnF sequences. Acta Botanica Boreali-Occidentalia Sinica， 2014， 34（2）： 265-269.
	狄红艳，骆凯，张吉宇，等. 基于ITS和trnL-trnF序列的草木樨种群遗传多样性研究. 西北植物学报， 2014， 34（2）： 265-269.
20	Wu F， Luo K， Yan Z Z， et al. Analysis of miRNAs and their target genes in five Melilotus albus NILs with different coumarin content. Scientific Reports， 2018， 8（1）： 1-13.
21	Wang P L， Yan Z Z， Gao L J， et al. Analysis of genetic variation in agronomic traits of half-sib families of Melilotus albus in the second generation of recurrent selection. Acta Prataculturae Sinica， 2022， 31（1）： 238-245.
	王朋磊，剡转转，高莉娟，等. 白花草木樨第二次轮回选择半同胞家系农艺性状的遗传变异分析. 草业学报， 2022， 31（1）： 238-245.
22	Wang S S， Duan Z， Zhang J Y. Establishment of hairy root transformation system of Melilotus albus induced by Agrobacterium rhizogenes. Acta Agrestia Sinica， 2021， 29（11）： 2591-2599.
	王升升，段珍，张吉宇. 发根农杆菌介导的白花草木樨毛状根转化体系的建立. 草地学报， 2021， 29（11）： 2591-2599.
23	Luo K， Jahufer M Z Z， Zhao H， et al. Genetic improvement of key agronomic traits in Melilotus albus. Crop Science， 2018， 58（1）： 285-294.
24	Chen L J， Wang P L， Chen X M， et al. Recurrent selection of new breeding lines and yield potential， nutrient profile and in vitro rumen characteristics of Melilotus officinalis. Field Crops Research， 2022， 287： 108657.
25	Utrup L J， Cary A J， Norris J H. Five nodulation mutants of white sweetclover （Melilotus-alba Dser） exhibit distinct phenotypes blocked at root hair curling， infection thread development， and nodule organogenesis. Plant Physiology， 1993， 103（3）： 925-932.
26	Lum M R， Li Y， LaRue T A， et al. Investigation of four classes of non-nodulating white sweetclover （Melilotus alba annua Desr.） mutants and their responses to arbuscular-mycorrhizal fungi. Integrative and Comparative Biology， 2002， 42（2）： 295-303.
27	Cooper J E. Early interactions between legumes and rhizobia： Disclosing complexity in a molecular dialogue. Journal of Applied Microbiology， 2007， 103（5）： 1355-1365.
28	Chaintreuil C， Rivallan R， Bertioli D J， et al. A gene-based map of the nod factor-independent Aeschynomene evenia sheds new light on the evolution of nodulation and legume genomes. DNA Research， 2016， 23（4）： 365-376.
29	Saeki K. Rhizobial measures to evade host defense strategies and endogenous threats to persistent symbiotic nitrogen fixation： A focus on two legume-rhizobium model systems. Cellular and Molecular Life Sciences， 2011， 68（8）： 1327-1339.
30	Ferguson B J， Indrasumunar A， Hayashi S， et al. Molecular analysis of legume nodule development and autoregulation. Journal of Integrative Plant Biology， 2010， 52（1）： 61-76.
31	Limpens E， Franken C， Smit P， et al. LysM domain receptor kinases regulating rhizobial nod factor-induced infection. Science， 2003， 302（5645）： 630-633.
32	Ane J M， Kiss G B， Riely B K， et al. Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes. Science， 2004， 303（5662）： 1364-1367.
33	Levy J， Bres C， Geurts R， et al. A putative Ca²⁺ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses. Science， 2004， 303（5662）： 1361-1364.
34	Hirsch S， Kim J， Munoz A， et al. GRAS proteins form a DNA binding complex to induce gene expression during nodulation signaling in Medicago truncatula. Plant Cell， 2009， 21（2）： 545-557.
35	Feng Y. Molecular mechanism of symbiosis receptor kinase regulating symbiosis nitrogen fixation in Lotus japonicus. Wuhan： Huazhong Agricultural University， 2020.
	冯勇. 百脉根共生受体激酶SymRK 调控共生固氮的分子机制研究. 武汉：华中农业大学， 2020.
36	Wu F， Duan Z， Xu P， et al. Genome and systems biology of Melilotus albus provides insights into coumarins biosynthesis. Plant Biotechnology Journal， 2022， 20（3）： 592-609.
37	Borisov A Y， Madsen L H， Tsyganov V E， et al. The sym35 gene required for root nodule development in pea is an ortholog of Nin from Lotus japonicus. Plant Physiology， 2003， 131（3）： 1009-1017.

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