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. 2022 Mar;28(3):573-583.
doi: 10.1007/s12298-022-01161-z. Epub 2022 Mar 29.

A novel SCARECROW-LIKE3 transcription factor LjGRAS36 in Lotus japonicus regulates the development of arbuscular mycorrhizal symbiosis

Yunjian Xu  1   2   3 , Fang Liu  3   4 , Fulang Wu  3 , Manli Zhao  3 , Ruifan Zou  3 , Jianping Wu  1   2 , Xiaoyu Li  3
Affiliations

A novel SCARECROW-LIKE3 transcription factor LjGRAS36 in Lotus japonicus regulates the development of arbuscular mycorrhizal symbiosis

Yunjian Xu et al. Physiol Mol Biol Plants. 2022 Mar.

Abstract

The symbiosis with arbuscular mycorrhizal (AM) fungi improves plants' nutrient uptake. During this process, transcription factors have been highlighted to play crucial roles. Members of the GRAS transcription factor gene family have been reported involved in AM symbiosis, but little is known about SCARECROW-LIKE3 (SCL3) genes belonging to this family in Lotus japonicus. In this study, 67 LjGRAS genes were identified from the L. japonicus genome, seven of which were clustered in the SCL3 group. Three of the seven LjGRAS genes expression levels were upregulated by AM fungal inoculation, and our biochemical results showed that the expression of LjGRAS36 was specifically induced by AM colonization. Functional loss of LjGRAS36 in mutant ljgras36 plants exhibited a significantly reduced mycorrhizal colonization rate and arbuscular size. Transcriptome analysis showed a deficiency of LjGRAS36 led to the dysregulation of the gibberellic acid signal pathway associated with AM symbiosis. Together, this study provides important insights for understanding the important potential function of SCL3 genes in regulating AM symbiotic development.

Supplementary information: The online version contains supplementary material available at 10.1007/s12298-022-01161-z.

Keywords: Arbuscular mycorrhizal fungi; Gibberellic acid signal pathway; Lotus japonicus; SCL3; Symbiosis.

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Conflict of interest statement

Conflict of interestThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Phylogenetic analysis of LjGRAS members. Symbiosis-related GRASs were indicated by solid green blocks. GRAS proteins from Arabidopsis (At), maize (Zm), rice (Os), Medicago truncatula (Mt), Lotus japonicus (Lj), Brassica napus (Bn), Brassica rapa (Br), Populus trichocarpa (Pt), Ricinus communis (Rc), Sorghum bicolor (Sb), Setaria italica (Si), Solanum lycopersicum (Sl), Hordeum vulgare (Hv), Triticum aestivum (Ta), Solanum tuberosum (St) and Theobroma cacao (Tc). The phylogenetic tree was generated by MEGA6. Neighbor-Joining model (bootstrap = 1000) was used
Fig. 2
Fig. 2
Heat map representation and hierarchical clustering of SCL3 members in different tissues. Data were obtained from Lotus base (https://lotus.au.dk/). The value bar was shown on the right, and red to blue colors represent high to low expression levels
Fig. 3
Fig. 3
Expression patterns of SCL3 members in response to AM symbiosis. Data were obtained from Lotus base (https://lotus.au.dk/). Amnon represents roots not inoculated with AM fungi and AM27 represents roots inoculated with AM fungi for 27 days. Error bars represent SD. Asterisks indicate significant differences between non-colonized and colonized roots (Student’s t-test, *P < 0.05 and **P < 0.01)
Fig. 4
Fig. 4
Histochemical staining for the promoter activity of LjGRAS36 using the GUS reporter gene. (A) AM-responsive elements analysis of LjGRAS36 promoter by RSAT. (B) GUS activity was not detected in the roots of pLjGRAS36:GUS transgenic plants in absence of R. irregularis. Bar = 200 μm. (C) GUS activity was detected in the roots of pLjGRAS36:GUS transgenic plants in presence of R. irregularis. Bar = 200 μm
Fig. 5
Fig. 5
Mycorrhizal phenotype in ljgras36 mutant. (A) Expression levels of LjGRAS36 in R. irregularis colonized (+ AMF) and non-colonized (-AMF) roots of wild type (WT) and ljgras36. Three biological replicates were used. Error bars represent SD. Different letters above the columns indicate significant differences at the P < 0.05 level. (B) Micrograph of mycorrhizal ljgras36 mutant and WT. Bar = 500 μm. (C) Mycorrhization rate of R. irregularis colonized roots of ljgras36 and WT at four weeks post-inoculation. Error bars represent SD. Three biological replicates with approximately 90 root segments per genotype were set. Asterisks indicate significant differences between WT and ljgras36 (Student’s t-test, *P < 0.05). (D) Expression levels of RiLSU in R. irregularis colonized roots of wild type (WT) and ljgras36. RiLSU indicates R. irregularis large subunit. Three biological replicates were used. Error bars represent SD. Asterisks indicate significant differences between WT and ljgras36 mutant (Student’s t test, *P < 0.05). (E) Arbuscular size classes in the WT and ljgras36 mutant plants. Three biological replicates with approximately 400 arbuscules were measured per genotype. Error bars represent SD. Asterisks indicate significant differences between WT and ljgras36 mutant (Student’s t-test, *P < 0.05)
Fig. 6
Fig. 6
LjGRAS36 influences on the root of Lotus japonicus post-AMF colonization. (A) Phenotypes of wild type L. japonicus and ljgras36 mutant post AM colonization. Bar = 2 cm. (B) Root length of WT and ljgras36 mutant post-AM colonization (Student’s t-test, *P < 0.05). Seventeen plants were measured for each phenotype. (C) Hierarchical cluster analysis of the differentially expressed genes (DEGs, log2(fold change) > 1 or log2(fold change) < -1, p < 0.05) between roots of wild type (WT) and ljgras36 post AM fungi colonization. +AMF means roots colonized by AM fungi. (D) Pie chart presentation of KEGG pathway analysis between WT and ljgras36
Fig. 7
Fig. 7
Expression patterns of DEGs related to GA signal transduction pathway in wild type post AM colonization. The expression date was collected from Lotus Base (https://lotus.au.dk/). AMnon represents roots that were not inoculated with AM fungi, and AM27 represents roots that were inoculated with AM fungi for 27 days
Fig. 8
Fig. 8
Hypothesized model of the mechanism that LjGRAS36 regulates arbuscular development
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