Plant Molecular Biology

, Volume 96, Issue 4–5, pp 473–492 | Cite as

Analysis of physiological and miRNA responses to Pi deficiency in alfalfa (Medicago sativa L.)

  • Zhenyi Li
  • Hongyu Xu
  • Yue Li
  • Xiufu Wan
  • Zhao Ma
  • Jing Cao
  • Zhensong Li
  • Feng He
  • Yufei Wang
  • Liqiang Wan
  • Zongyong Tong
  • Xianglin Li


Key message

The induction of miR399 and miR398 and the inhibition of miR156, miR159, miR160, miR171, miR2111, and miR2643 were observed under Pi deficiency in alfalfa. The miRNA-mediated genes involved in basic metabolic process, root and shoot development, stress response and Pi uptake.


Inorganic phosphate (Pi) deficiency is known to be a limiting factor in plant development and growth. However, the underlying miRNAs associated with the Pi deficiency-responsive mechanism in alfalfa are unclear. To elucidate the molecular mechanism at the miRNA level, we constructed four small RNA (sRNA) libraries from the roots and shoots of alfalfa grown under normal or Pi-deficient conditions. In the present study, alfalfa plants showed reductions in biomass, photosynthesis, and Pi content and increases in their root-to-shoot ratio and citric, malic, and succinic acid contents under Pi limitation. Sequencing results identified 47 and 44 differentially expressed miRNAs in the roots and shoots, respectively. Furthermore, 909 potential target genes were predicted, and some targets were validated by RLM-RACE assays. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed prominent enrichment in signal transducer activity, binding and basic metabolic pathways for carbohydrates, fatty acids and amino acids; cellular response to hormone stimulus and response to auxin pathways were also enriched. qPCR results verified that the differentially expressed miRNA profile was consistent with sequencing results, and putative target genes exhibited opposite expression patterns. In this study, the miRNAs associated with the response to Pi limitation in alfalfa were identified. In addition, there was an enrichment of miRNA-targeted genes involved in biological regulatory processes such as basic metabolic pathways, root and shoot development, stress response, Pi transportation and citric acid secretion.


Low inorganic phosphate Medicago sativa Physiological indexes MicroRNA Deficient-Pi-responsive genes 



This work was supported by China Forage and Grass Research System (CARS-34) , and the Agricultural Science and Technology Innovation Program (ASTIP-IAS14).

Author contributions

ZL, XL and ZT conceived and designed the experiments. ZL, HX, YL, JC, XW, ZL and ZM performed the experiments. ZL analyzed the experimental data and drafted the manuscript. FH, YW and LW revised the draft manuscript. XL and ZT supervised all analyses and revised the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11103_2018_711_MOESM1_ESM.tif (41.4 mb)
Supplementary Fig. 1 Flow diagram for the analysis of small RNAs. Supplementary material 1 (TIF 42397 KB)
11103_2018_711_MOESM2_ESM.tif (28.4 mb)
Supplementary Fig. 2 The distribution of different small RNAs in different chromosome. Supplementary material 2 (TIF 29101 KB)
11103_2018_711_MOESM3_ESM.tif (56 mb)
Supplementary Fig. 3 Percent nucleotide bias of the 20th -24th bases of novel miRNAs from 12 libraries. The brown box indicates guanine, the green box indicates cytosine, the blue box indicates uracil, and the red box indicates adenine. Supplementary material 3 (TIF 57371 KB)
11103_2018_711_MOESM4_ESM.tif (54.4 mb)
Supplementary Fig. 4 Secondary structures of predicted novel miRNAs. Supplementary material 4 (TIF 55753 KB)
11103_2018_711_MOESM5_ESM.tif (41.6 mb)
Supplementary Fig. 5 Pearson correlation of miRNA expression between different libraries. Blue indicates that the correlation coefficient is 1. Supplementary material 5 (TIF 42593 KB)
11103_2018_711_MOESM6_ESM.tif (13.2 mb)
Supplementary Fig. 6 Common and specific differentially expressed miRNAs in roots and shoots under Pi-deficient conditions. (a) Conserved miRNA response to Pi deficiency; (b) Novel miRNA response to Pi deficiency. Supplementary material 6 (TIF 13509 KB)
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Supplementary material 7 (DOCX 22 KB)
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Supplementary material 8 (XLSX 14044 KB)
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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Zhenyi Li
    • 1
  • Hongyu Xu
    • 1
  • Yue Li
    • 1
  • Xiufu Wan
    • 2
  • Zhao Ma
    • 1
  • Jing Cao
    • 1
  • Zhensong Li
    • 1
  • Feng He
    • 1
  • Yufei Wang
    • 1
  • Liqiang Wan
    • 1
  • Zongyong Tong
    • 1
  • Xianglin Li
    • 1
  1. 1.Institute of Animal SciencesChinese Academy of Agricultural SciencesBeijingChina
  2. 2.State Key Laboratory of Dao-Di Herbs, National Resource Center for Chinese MedicaChina Academy of Chinese Medical SciencesBeijingChina

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