Effects of Nitrogen Application on Nitrogen Fixation Capacity and GmLbs Expression in Soybean

doi:10.11983/CBB21037

Abstract

Abstract: The nitrogen fixation efficiency of symbiotic nodules is tightly regulated by external nitrogen (N). In addition to nitrogenase activity, the leghemoglobin (Lb) amount is vital index contributing to N₂fixation. To determine the effect of environmental N level on biological nitrogen fixation, soybean plants were inoculated with rhizobia for 30 d under low N condition (0.53 mmol·L^-1), then transplanted to hydroponic culture solution with relatively higher N concentrations, including 5.3, 10, 20, 30, and 40 mmol·L^-1 for 7 d, respectively. Lb concentration, nitrogenase activity and bacteroid development status were measured. Nodule displayed changed color from red to green with increasing N concentrations. Consistently, the red Lb concentration gradually declined with increased green Lb concentration. Moreover, the nitrogenase activity, infected cell number and area in nodules were all significantly decreased, suggesting that changes in Lb forms caused by excess N are closely associated with nitrogen fixation capacity. Bioinformatics and public expression profile data displayed that four symbiosis-associated Lbs, including GmLb1, GmLb2, GmLb3, and GmLb4 are the major Lb genes in soybean nodulation. These four GmLbs belong to the same clade of phylogenetic tree. Further analysis on the transcripts of GmLb1-4 in response to N showed that the expression levels of GmLb1-4 were all significantly inhibited by high N. This study services as a reference for future studies in understanding the underlying mechanisms of N-triggered nodule senescence as well as BNF application in agriculture.

Key words: biological nitrogen fixation, GmLbs, leghemoglobin, nitrogen concentration, soybean

Mengke Du, Wenting Lian, Xiao Zhang, Xinxin Li. Effects of Nitrogen Application on Nitrogen Fixation Capacity and GmLbs Expression in Soybean[J]. Chinese Bulletin of Botany, 2021, 56(4): 391-403.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB21037

https://www.chinbullbotany.com/EN/Y2021/V56/I4/391

Figures/Tables 8

Table 1 Gene-specific primers used for qRT-PCR analysis

Name	Primer sequence (5'-3')
GmLb1-qF	CCTCGATACTGGAGAAAGCACC
GmLb1-qR	CAAGTGCGGCATCAATCACC
GmLb2-qF	AATGGAACAGTGGTGGCTGA
GmLb2-qR	AGCACTGCTCAATTCGTCAC
GmLb3-qF	CCGCACTTGGTTCTGTTCAT
GmLb3-qR	TGCTGCCAATTCATCGTAGG
GmLb4-qF	GATCTACTATTGCCGTCAA
GmLb4-qR	GCATCGATTATGATTCACA

Figure 1 Effects of nitrogen supply on leghemoglobin (Lb) concentration and nitrogenase activity of soybean nodules (A) Cross section of nodules; (B) Leghemoglobin concentration; (C) Nitrogenase activity. There were 4 biological replicates, 2 or 3 nodules were randomly harvested for Lb concentration and nitrogenase activity analysis. Data are means±SE from 10 nodules. Different lowercase letters indicate significant differences among different N supply levels (P<0.05). Bars=2 mm

Figure 2 Effects of nitrogen supply on the infected cell development in nodules (A) Toluidine blue-stained nodule cross-sections; (B) Number of infected cells; (C) Surface area of infected cells. Data are means±SE from 40 cross of nodules. Different lowercase letters indicate significant differences among different N supply levels (P<0.05). Bars=200 µm

Table 2 Information of GmLbs/GmHbs genes in soybean

Gene name	Phytozome locus9.1	Phytozome locus12.0	NCBI number	Accession number	Location coordinates (5'-3')
GmLb1	Glyma10g34260	Glyma.10G198800.1	NM_001248494.2	BT092230	42996028-42997092
GmLb2	Glyma10g34280	Glyma.10G199000.1	NM_001358072.1	FK026737	43004554-43005947
GmLb3	Glyma10g34290	Glyma.10G199100.1	NM_001248999.3	BT092268	43009363-43011021
GmLb4	Glyma20g33290	Glyma.20G191200.1	NM_001248319.3	BT092218	42993081-42994203
GmLb5	Glyma10g34275	Glyma.10G198900.1	-	-	43000045-43003378
GmHb1	Glyma11g12960	Glyma.11G121700.1	NM_001255274.2	BT098807	9299437-9300752
GmHb2	Glyma11g12980	Glyma.11G121800.1	NM_001357481.1	BT096529	9303967-9305430

Figure 3 Prediction of the conserved domain and 3D protein structure of leghemoglobin (A) Prediction of the conserved domains (the yellow font represents the heme binding site, the black font represents the cofactor binding site, and the white font represents polypeptide binding site); (B) Prediction of 3D protein structure

Figure 4 Phylogenetic tree of hemoglobin This phylogenetic tree was constructed by the neighbor-joining method in the MEGA 6.1 program.

Figure 5 Heatmaps of GmLbs and GmHbs gene expression in soybean (A) The RNA-seq data obtained from Soybase website (https://www.soybase.org); (B) The RNA-seq data obtained from Libault et al. (2010). RNA-seq data of different sources were calculated with log10(FPKM+1) and further used to generate the heatmaps. DAF: Days after flowering; HAS: Hours after sowing; SAM: Shoot apical meristem

Figure 6 Relative expression value of GmLb1-4 in soybean nodules under different nitrogen concentrations Data are means±SE of 3 biological replicates.

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