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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (2): 353-366.doi: 10.3724/SP.J.1006.2022.14006


Identification, expression profile of soybean PIN-Like (PILS) gene family and its function in symbiotic nitrogen fixation in root nodules

DONG Yan-Kun1(), HUANG Ding-Quan2, GAO Zhen2, CHEN Xu2,*()   

  1. 1College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    2Haixia Institute of Science and Technology, Horticultural Plant Biology and Metabolomics Center, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
  • Received:2021-01-11 Accepted:2021-04-26 Online:2022-02-12 Published:2021-05-18
  • Contact: CHEN Xu E-mail:dongyk1124@163.com;chenxu@inbox.com
  • Supported by:
    This study was supported by the National Key Research and Development Program of China “Seven Major Crop Breeding” Special Topic Crop Organ Development and Nutrient Efficient Use Interaction Mechanism(2016YFD0100705)


Plant hormone auxin plays a vital role in the growth and development of plants. Auxin homeostasis and concentration gradient establishment control the polar formation of almost all organs. The synthesis, transportation, perception, and metabolic degradation of auxin in specific cells establish a concentration gradient of auxin in accordance with organ development. In legumes, roots interact with soil microorganisms to form a special organ called nodules, which is used for biological nitrogen fixation. However, the function of auxin homeostasis control of biological nitrogen fixation is unknown. Studies showed that PIN-Like (PILS) proteins in Arabidopsis helped to regulate intracellular auxin homeostasis and mediate auxin signal transmission in the downstream nucleus. In this study, 19 PILS family genes (GmPILSs) were identified in soybean genome and distributed unevenly on 10 chromosomes of soybean. GmPILSs exhibited a variety of expression patterns in nine tissue parts of soybean, and had obvious specificity of tissue expression. GmPILS1e and GmPILS1f were enriched and expressed in the rhizobia region, and the expression of GmPILS1e and GmPILS1f in nodules was down-regulated by artificial microRNA interference (amiRNAi), resulting in the increase of nitrogenase activity in the nodules. However, the overexpression of GmPILS1f leaded to the decrease nitrogenase activity in root nodules, GmPILS1e and GmPILS1f might participate in the regulation of soybean nitrogenase activity. These results lay the foundation for further analysis of the function and mechanism of soybean GmPILS family genes, and also provide valuable genetic resources for the application of nodulation and nitrogen fixation in agricultural breeding.

Key words: Glycine max, PIN-Like (PILS) gene family, nodule, symbiotic nitrogen fixation

Table 1

Basic information of GmPILS gene family"

Gene name
Gene ID
No. of amino acids
No. of introns
5'-3' corrdinates
GmPILS1a Glyma.10G189100 313 6 Chr10: 42227422-42231792
GmPILS1b Glyma.20G201600 400 10 Chr20: 43857079-43862374
GmPILS1c Glyma.10G189000 400 10 Chr10: 42220046-42225237
GmPILS1d Glyma.20G201700 259 8 Chr20: 43863679-43868413
GmPILS1e Glyma.07G113100 418 9 Chr07: 11724201-11741130
GmPILS1f Glyma.07G113200 418 11 Chr07: 11780634-11792223
GmPILS1g Glyma.03G113600 424 10 Chr03: 32066847-32080424
GmPILS1h Glyma.16G114900 297 6 Chr16: 25456379-25485759
GmPILS1i Glyma.11G088600 415 10 Chr11: 6695030-6700783
GmPILS1j Glyma.01G156200 415 9 Chr01: 49361352-49366442
GmPILS1k Glyma.09G195600 414 10 Chr09: 42022526-42029030
GmPILS1l Glyma.16G115500 414 9 Chr16: 25622503-25630816
GmPILS2a Glyma.09G116100 440 1 Chr09: 26549730-26551878
GmPILS2b Glyma.19G072900 445 1 Chr19: 26101897-26103958
GmPILS5a Glyma.11G087300 419 10 Chr11: 6547702-6555660
GmPILS5b Glyma.01G157700 419 10 Chr01: 49570825-49577993
GmPILS5c Glyma.09G196900 409 9 Chr09: 42160775-42168544
GmPILS6a Glyma.09G271100 414 10 Chr09: 48791789-48796501
GmPILS6b Glyma.18G218300 414 10 Chr18: 50525980-50531206

Fig. 1

Gene structures of GmPILS gene family The yellow boxes represent the exons and black lines represent the introns of different GmPILS genes."

Fig. 2

Distribution of GmPILS genes on soybean chromosomes"

Fig. 3

Phylogenetic tree of GmPILS, AtPILS, and OsPILS proteins"

Fig. 4

Relative expression pattern of GmPILSs genes A: the heat map of tissue expression pattern of GmPILS genes, and FPKM is obtained from Phytozome database. B: GmPILS1e, GmPILS1f, GmPILS1i, GmPILS1j, and GmPILS5a gene tissue expression pattern in heat map; GmPILSs expression data comes from qPCP results. Colors in square represent the expression levels of GmPILSs."

Fig. 5

Histochemical localization of GmPILS1e, GmPILS1f, GmPILS1i, GmPILS1j, and GmPILS5a in nodule The figures from left to right are referred to the initiation, expansion, and maturity stages of nodules development. Bar: 200 µm."

Fig. 6

Subcellular localization of GmPILS1e and GmPILS1f A-D: tobacco leaves were used for co-transformation of the mixed bacterial broth of pro35S:GFP-GmPILS (green) or pro35S:GmPILS-GFP (green) and pro35S:HDEL-Tdtoamto (red, as an endoplasmic reticulum localization marker). The co-localization signal profile chart is generated based on the white slash. Bar: 10 μm."

Fig. 7

GmPILS1e and GmPILS1f were involved in the regulation of nitrogenase activity in soybean nodules A-G: phenotype analysis was performed on soybean complex plants after inoculation of rhizobia for 21 days. A: mock, GmPILS1e 1f-RNAi#1 and GmPILS1e 1f-RNAi#2 nodules by semi-crosscutting, bar: 200 µm. B: RT-qPCR analysis of GmPILS1e or GmPILS1f in mock, GmPILS1e 1f-RNAi#1 and GmPILS1e 1f-RNAi#2 nodules, with GmELF1b gene as the internal reference gene. C, D: test the nitrogenase activity of mock, GmPILS1e 1f-RNAi#1 and GmPILS1e 1f-RNAi#2 nodules of the same weight. E: statistic mock, GmPILS1e 1f-RNAi#1 and GmPILS1e 1f-RNAi#2. F: RT-qPCR analysis of GmPILS1f in Mock and 35S::GmPILS1f nodules, with GmELF1b gene as the internal reference gene. G: test the nitrogenase activity of mock and 35S::GmPILS1f nodules. *, **, and *** mean significant difference at the 0.05, 0.01, 0.001 probability levels, respectively. ns: no significant difference."

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