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Phylogenetic and Expression Analyses of Cullin Family Members Unveil the Role of PbCUL1.C1 in Pollen Tube Growth Underlying Non-self S-RNase in Pear

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Abstract

Cullin (CUL) gene family organizes the largest class of RING E3 ligases which play crucial roles in the ubiquitin-proteasome system to affect plant physiological activities. However, little is known about the CUL genes in fruit trees. In this study, 57 CUL genes were isolated from the sequenced genomes of seven Rosaceae trees and were divided into three subfamilies according to phylogenetic analyses. Evolutionary analysis indicated that multiple duplication events play important role in the expansion of the CUL gene family in Rosaceae. The expression pattern shows that only four CUL genes were highly expressed in pollen and pollen tube in pear. Of these CUL genes, PbCUL1.C1 presented the highest levels of expression in pollen, but the knockdown of PbCUL1.C1 by antisense oligonucleotide (ASO) did not influence pollen tube growth. Interestingly, the pollen tube treated by both the non-self S-RNase and ASO of PbCUL1.C1 was longer than that treated by unique non-self S-RNase, suggesting that the PbCUL1.C1 plays an important role for pollen tube growth underlying non-self S-RNase in pear. These results will be useful for exploring the biological roles of CUL genes in self-incompatibility in Rosaceae trees.

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Abbreviations

CUL :

Cullin

ASO:

antisense oligonucleotide

CRLs:

cullin-RING ligase complexes

SLF:

S-locus F-box

SCF:

S-phase kinase-associated protein 1 (SKP1)-CUL1 (CUL1)-F-box-RING box 1 (RBX1)

HMM:

Hidden Markov model

RT-PCR:

real-time PCR

RNA-Seq:

RNA sequencing

MP:

mature pollen grains

HP:

hydrated pollen grains

PT:

growing pollen tubes

SPT:

stopped-growth pollen tubes

qRT-PCR:

quantitative real-time PCR

WGD:

whole-genome duplication

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Funding

This work was financially supported by the National Natural Science Foundations of China (31772276 and 31672118).

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Authors and Affiliations

  1. Centre of Pear Engineering Technology Research, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, 210095, China

    Ye Zhou, Yongdan Huang, Lei Wu, Guoming Wang, Chao Gu & Shaoling Zhang

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  1. Ye Zhou
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  2. Yongdan Huang
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  3. Lei Wu
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  4. Guoming Wang
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  5. Chao Gu
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  6. Shaoling Zhang
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Contributions

SLZ and CG conceived and designed the experiments. YZ wrote the manuscript. YZ, YDH, LW, GMW conducted all the experiments and data analysis. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Chao Gu or Shaoling Zhang.

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Key Message

1. Fifty-seven CUL genes were identified from pear and other rosaceae fruit trees and can be phylogenetic classified into three groups;

2. Pear PbCUL1.C1 is expressed in all the tested tissues and more highly expressed in pollen tube;

3. The down-regulation of PbCUL1.C1 enhances pollen tube growth underlying non-self S-RNase.

Electronic supplementary material

Fig S1

The yeast two-hybrid assay. Full-length sequences of PbCUL1.C1 were amplified and inserted into vector PGBKT7. Full-length sequences of PbSSK1 and PbSSK2 were amplified and inserted into vector PGADT7. The LiC1-PEG method was used to co-transform the vectors into yeast strain AH109 cells. The transformants were selected on SD/Leu/-Trp medium. The interaction was detected on SD/-Leu/-Trp/-His/-Ade medium. The transformation of PGADT7-T and PGBKT7-Lam, PGADT7-T, and PGBKT7-53 were used as negative and positive controls. (PDF 487 kb)

Table S1

Primer pairs for CUL genes used for quantitative real-time PCR analysis. (XLSX 11 kb)

Table S2

The primer sequences used for ASO. (XLSX 10 kb)

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Zhou, Y., Huang, Y., Wu, L. et al. Phylogenetic and Expression Analyses of Cullin Family Members Unveil the Role of PbCUL1.C1 in Pollen Tube Growth Underlying Non-self S-RNase in Pear. Plant Mol Biol Rep 38, 601–612 (2020). https://doi.org/10.1007/s11105-020-01221-2

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