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Phylogeny and Evolution of Calcineurin B-Like (CBL) Gene Family in Grass and Functional Analyses of Rice CBLs

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Abstract

Calcium signals play critical functions in regulating diverse arrays of plant growth and development and mediating a variety of biotic and abiotic stress responses as a second messenger. Calcineurin B-like (CBL) proteins were involved with plant-specific Ca2+signaling as calcium sensors. In this work, we retrieved 152 CBL gene members from 15 different grass species, surveyed their phylogenetic relationships and sequence features and also performed expression patterns and functional analyses of rice CBLs. Phylogenetic analysis indicated that grass CBLs fall into four different groups (Group A–D). Sequence analysis showed that CBL proteins harboring four conserved calcium-binding EF-hand have key amino acid residues Asp and Glu which had relatively high proportion in the average abundance. Molecular evolutionary analyses revealed that group A, B and C CBLs in their evolution process suffered the purifying selection, while group D CBLs were subjected to positive selection. Moreover, expression analyses showed significant divergent expression patterns of OsCBLs in various organs and under different hormones and abiotic stresses. Furthermore, tolerance analysis revealed that OsCBL3 and OsCBL8 overexpression transgenic rice seedlings improved salt tolerance and OsCBL5, OsCBL6 and OsCBL7 positively regulated drought stress. In general, the domain and base sequence of the CBL gene family is highly conserved in grasses. OsCBL genes had specific gene expression profiles and function in different stresses.

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Abbreviations

CBL:

Calcineurin B-like

[Ca2+]cyt:

Cytosolic calcium concentration

EF-hand:

Elongation factor hand

CaM:

Calmodulin

CMLs:

CaM-like proteins

CDPKs:

Ca2+-dependent protein kinases

CIPKs:

CBL-interacting protein kinases

CNB:

Calcineurin B

AKT1:

Arabidopsis K+ transporter 1

pI:

Isoelectric point

Mw:

Molecular weight

ML:

Maximum-likelihood

JTT:

Jones-Taylor-Thornton

ZH11:

Zhonghua 11

RT-qPCR:

Real-time quantitative PCR

SAM:

Shoot apical meristem

MS:

Murashige and Skoog

WT:

Wild type

ώ = dN/dS :

Nonsynonymous-to-synonymous rates ratio

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Funding

This research was funded by the National Natural Science Foundation of China (81360611) and Shanghai Sailing Program (19YF1414800). The funding bodies had no role in study design, analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Research and Development Center for Heath Product, College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, 650201, Yunnan, China

    Min Jiang, Changling Zhao, Mingfu Zhao, Yongzhong Li & Guosong Wen

  2. Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences(CAS), Shanghai Chenshan Botanic Garden, Shanghai, 201602, China

    Min Jiang

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  1. Min Jiang
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  2. Changling Zhao
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  3. Mingfu Zhao
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  4. Yongzhong Li
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  5. Guosong Wen
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Contributions

MJ and GW conceived and designed the project. MJ executed the bioinformatics analysis, conducted the experiments and wrote the manuscript. CZ, MZ, YL and GW retrieved gene sequence data and performed the bioinformatics analysis. All authors read and approved the final manuscript.

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Correspondence to Guosong Wen.

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Supplementary file1 (DOCX 17 kb)

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12374_2020_9240_MOESM7_ESM.tif

Fig. S1. Maximum Likelihood phylogenetic trees were reconstructed using EF-hand motifs of CBL proteins in 15 grasses. Phylogenetic analysis was carried with EF-hand motifs of protein sequences for 152 CBL proteins from 15 grass species identified in this study Supplementary file7 (TIF 8180 kb)

12374_2020_9240_MOESM8_ESM.tif

Fig. S2. Sequence features shown in the form of web logos representing the conserved EF-hand motif of each group. The red stars indicate residues of functional or structural importance based on phylogenetic conservations. Logos were generated using the Weblogo3 application (http://weblogo.threeplusone.com/) Supplementary file8 (TIF 4225 kb)

12374_2020_9240_MOESM9_ESM.tif

Fig. S3. Sequence features shown in the form of web logos representing the conserved FPSF domain in C-terminal region of each group Supplementary file9 (TIF 1744 kb)

12374_2020_9240_MOESM10_ESM.tif

Fig. S4. Molecular analysis of OsCBLs transgenic rice plants in the level of DNA. 1: OsCBL1,2: OsCBL2,3: OsCBL3,4: OsCBL4,5: OsCBL5,6: OsCBL6,7: OsCBL7,8: OsCBL8,9: OsCBL9,10: OsCBL10, Marker:100 bp plus DNA ladder Supplementary file10 (TIF 2635 kb)

Fig. S5. GUS staining of OsCBLs transgenic and WT rice plants Supplementary file11 (TIF 6689 kb)

12374_2020_9240_MOESM12_ESM.tif

Fig. S6. Real-time PCR analysis of OsCBLs T3 transgenic rice lines. A: OsCBL1-OE, B:OsCBL2-OE, C:OsCBL3-OE, D: OsCBL4-OE, E: OsCBL5-OE, F: OsCBL6-OE, G: OsCBL7-OE, H:OsCBL8-OE, I: OsCBL9-OE, J:OsCBL10-OE Supplementary file12 (TIF 7048 kb)

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Jiang, M., Zhao, C., Zhao, M. et al. Phylogeny and Evolution of Calcineurin B-Like (CBL) Gene Family in Grass and Functional Analyses of Rice CBLs. J. Plant Biol. 63, 117–130 (2020). https://doi.org/10.1007/s12374-020-09240-y

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