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Functional and transcriptomic analyses of the NF-Y family provide insights into the defense mechanisms of honeybees under adverse circumstances

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Abstract

As predominant pollinators, honeybees are important for crop production and terrestrial ecosystems. Recently, various environmental stresses have led to large declines in honeybee populations in many regions. The ability of honeybees to respond to these stresses is critical for their survival. However, the details of the stress defense mechanisms of honeybees have remained elusive. Here, we found that the Nuclear Factor Y (NF-Y) family (containing NF-YA, NF-YB, and NF-YC) is a novel stress mediator family that regulates honeybee environmental stress resistance. NF-YA localized in the nucleus, NF-YB accumulated in the cytoplasm, and NF-YC presented in both the nucleus and cytoplasm. NF-YC interacted with NF-YA and NF-YB in vitro and in vivo, and the nuclear import of NF-YB relied on its interaction with NF-YC. We further found that the expression of NF-Y was induced under multiple stress conditions. In addition, NF-Y regulated many stress responses and antioxidant genes at the transcriptome-wide level, and knockdown of NF-Y repressed the expression of stress-inducible genes, particularly LOC108003540 and LOC107994062, under adverse circumstances. Silencing NF-Y lowered honeybee stress resistance by reducing total antioxidant capacity and enhancing oxidative impairment. Collectively, these results indicate that NF-Y plays important roles in stress responses. Our study sheds light on the underlying defense mechanisms of honeybees under environmental stress.

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Acknowledgements

We thank Dr. Martin Giurfa, from Research Centre on Animal Cognition, University of Toulouse, and Dr. Panuwan Chantawannakul, from Bee Protection Laboratory, Chiang Mai University, for critical reading.

Funding

This work was financially supported by the Funds of Shandong Province “Double Tops” Program, the Shandong Province Modern Agricultural Technology System Innovation Team Special Fund (No. SDAIT-24-04), the National Natural Science Foundation of China (No. 31572470), and the Earmarked Fund for the China Agriculture Research System (No. CARS-44).

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Author notes

  1. Guilin Li and Hang Zhao have contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian, 271018, Shandong, People’s Republic of China

    Guilin Li, Hang Zhao & Xingqi Guo

  2. College of Animal Science and Technology, Shandong Agricultural University, Taian, 271018, Shandong, People’s Republic of China

    Ying Wang, Xuepei Cui & Baohua Xu

  3. Statistics Department, University of Auckland, 38 Princes Street, Auckland, New Zealand

    Hongbin Guo

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  1. Guilin Li
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Contributions

XG and BX contributed to study conception and design. GL, HZ, and HG prepared the materials and carried out the experimental work. HG, YW, and XC collected and analyzed the data. The first draft of the manuscript was written by GL and HZ, and all authors commented on the various versions of the manuscript before publishing. All authors read and approved the final manuscript.

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Correspondence to Baohua Xu or Xingqi Guo.

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18_2019_3447_MOESM1_ESM.jpg

Fig. S1. Genomic structure comparison among NF-Y gene family members. Lengths of the genomic DNA sequences of honeybee (Apis cerana and Apis mellifera) NF-YA (A), NF-YB (B) and NF-YC (C) and their homologs in Tyto alba, Pieris rapae, Bos taurus and Cimex lectularius. The 5′UTRs, 3′UTRs, introns and exons of the NF-Ys are presented in various colored boxes. BtNF-YA, Bos Taurus NF-YA (NP_001014956.1). PrNF-YA, Pieris rapae NF-YA (XP_022116701.1). ClNF-YA, Cimex lectularius NF-YA (XP_014249448.1). TaNF-YA, Tyto alba NF-YA (KFV55528.1). AccNF-YA, Apis cerana cerana NF-YA (XP_016910727.1). AmNF-YA, Apis mellifera NF-YA (XP_001121566.1). BtNF-YB, Bos Taurus NF-YB (NP_001073254.1). PrNF-YB, Pieris rapae NF-YB (XP_022121715.1). ClNF-YB, Cimex lectularius NF-YB (XP_024083212.1). TaNF-YB, Tyto alba NF-YB (XP_009972647.1). AccNF-YB, Apis cerana cerana NF-YB (XP_016914525.1). AmNF-YB, Apis mellifera NF-YB (XP_394667.3). BtNF-YC, Bos Taurus NF-YC (NP_001029770.1). PrNF-YC, Pieris rapae NF-YC (XP_022119667.1). ClNF-YC, Cimex lectularius NF-YC (XP_014260208.1). TaNF-YC, Tyto alba NF-YC (KFV48877.1). AccNF-YC, Apis cerana cerana NF-YC (XP_016907622.1). AmNF-YC, Apis mellifera NF-YC (XP_392156.2). (JPG 995 kb)

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Fig. S2. Multiple alignment of the amino acid sequences of NF-YA (A), NF-YB (B) and NF-YC (C) among different species. The conserved domain of NF-Y is marked with a red box. Acc, Apis cerana cerana. Am, Apis mellifera. Bt, Bos taurus. Cl, Cimex lectularius. Pr, Pieris rapae. Ta, Tyto alba. The GenBank accession number of each protein used here can be found in Fig. 1. (JPG 4041 kb)

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Fig. S3. Expression levels of NF-Y in Apis mellifera under 47°C treatment from 0 h to 5 h (A) or under 40°C treatment from 0 h to 48 h (B) compared to normal conditions (33°C). β-actin was used as an internal control. The data are presented as the mean ± SD of three replicates of four individuals each. *P < 0.05 and **P < 0.01 by Student’s t test. (JPG 480 kb)

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Fig. S4. RNAi efficiency of NF-YB (A) and NF-YC (B) as detected by RNA-seq. The NF-YB knockdown, NF-YC knockdown and GFP control honeybees are abbreviated with nf-yb-1-, nf-yc-1- and gfp, respectively. The data are shown as the mean ± SD of three biological replicates. **P < 0.01 by Student’s t test. (JPG 116 kb)

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Fig. S5. Scatter plot showing the differentially expressed genes in NF-YB knockdown compared to control honeybees. The NF-YB knockdown and GFP control honeybees are abbreviated with nf-yb-1- and gfp, respectively. (JPG 610 kb)

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Fig. S6. Scatter plot showing the differentially expressed genes in NF-YC knockdown compared to control honeybees. The NF-YC knockdown and GFP control honeybees are abbreviated with nf-yc-1- and gfp, respectively. (JPG 548 kb)

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Fig. S7. Representative list of the genes regulated by NF-YB (left panel) and NF-YC (right panel) associated with environmental stress responses, immune processes, development and antioxidative defense. The genes upregulated and downregulated by NF-YB or NF-YC as revealed by RNA-seq analysis are indicated with upward red arrows and downward blue arrows, respectively. The NF-YB knockdown and NF-YC knockdown honeybees are abbreviated with nf-yb-1- and nf-yc-1 (JPG 1439 kb)

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Fig. S8. Confirmation of stress response gene coregulation by NF-YB and NF-YC using RT-qPCR. The data are presented as the mean ± SD, n = 3. *P < 0.05 and **P < 0.01 by Student’s t test. β-actin was used as an internal control. The NF-YB knockdown, NF-YC knockdown and GFP control honeybees are abbreviated with nf-yb-1-, nf-yc-1- and gfp, respectively (JPG 691 kb)

18_2019_3447_MOESM9_ESM.jpg

Fig. S9. Expression levels of some antioxidative defense genes coregulated by NF-YB and NF-YC as detected by RT-qPCR. β-actin was used as an internal control. The data are presented as the mean ± SD, n = 3. **P < 0.01 by Student’s t test. The NF-YB knockdown, NF-YC knockdown and GFP control honeybees are abbreviated with nf-yb-1-, nf-yc-1- and gfp, respectively. (JPG 686 kb)

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Fig. S10. Expression levels of stress response genes and antioxidant genes in dsRNA-NF-YB-2-fed honeybees (nf-yb-2-) and dsRNA-NF-YC-2-fed honeybees (nf-yc-2-) compared to dsRNA-GFP-fed honeybees (gfp). A, Schematic drawings showing the targeting sequences of dsRNA-NF-YB-1, dsRNA-NF-YB-2, dsRNA-NF-YC-1 and dsRNA-NF-YC-2 in the CDSs of NF-YB and NF-YC. HFM: histone fold motif. B, RNAi efficiency of NF-YB and NF-YC in nf-yb-2- and nf-yc-2- compared to gfp. The data are presented as the mean ± SD of three biological replicates. **P < 0.01 by Student’s t test. C, mRNA levels of select genes in nf-yb-2- and nf-yc-2- compared to gfp control. The data are shown as the mean ± SD of three biological replicates. **P < 0.05 and **P < 0.01 by Student’s t test. (JPG 1155 kb)

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Li, G., Zhao, H., Guo, H. et al. Functional and transcriptomic analyses of the NF-Y family provide insights into the defense mechanisms of honeybees under adverse circumstances. Cell. Mol. Life Sci. 77, 4977–4995 (2020). https://doi.org/10.1007/s00018-019-03447-0

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