A female in vivo haploid-induction system via mutagenesis of egg cell-specific peptidases

doi:10.21203/rs.3.rs-1340747/v1

PDF

Letter

A female in vivo haploid-induction system via mutagenesis of egg cell-specific peptidases

https://doi.org/10.21203/rs.3.rs-1340747/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted 15 Feb, 2022

You are reading this latest preprint version

Crop breeding schemes can be significantly accelerated by using (doubled) haploid plants. In vivo haploid induction has been applied in plant breeding since decades, but is still not available for all crops, genotypes and haploidization rates are very low1-3. Therefore, plant breeders are highly interested in methodological improvements, especially, in new concepts to haploidization. Here, we report a novel system for the induction of haploid plants by mutating genes encoding egg cell-specific aspartic endopeptidases (ECSs). After successful sperm-egg fusion, ECSs play a critical role to ensure male and female nucleus fusion and parental genome incorporation. The ecs1 ecs2 double mutant pollinated by wild type pollen is capable to produce haploid offspring originating from semigamous zygotes. Chromosome elimination occurs during zygote and early embryo development. In summary, we report a novel approach for haploidization and additionally provide new insights into the molecular basis of fertilization.

Plant materials and growth conditions. T-DNA insertion lines SALK_021086 (ecs1) and SALK_090795 (ecs2) were obtained from the Arabidopsis Biological Resource Center (ABRC). Arabidopsis thaliana Columbia-0 (Col-0) was used as wild type (WT) control. Plants were grown in soil in a greenhouse under long‐day conditions (16 h light/8 h dark) at 22 °C.

Vector construction and plant transformation. All fragments were amplified by polymerase chain reaction (PCR) using the Phanta Kit (Vazyme Biotech). Primer sequences (synthesized by Sangon Biotech) are listed in Supplementary Table 1. All constructs were generated using restriction enzymes (New England Biolabs) and One Step Cloning Kit (Vazyme Biotech). To label egg cell membrane and chromosomes, DD45::GFP-LTI6b and DD45::CENH3-GFP fusion constructs were generated^{17, 24}. A DNA fragment containing the promoter and coding sequence was amplified from A. thaliana ecotype Col-0 genomic DNA and inserted into the P094 vector. All constructs were transformed into Agrobacterium tumefaciens strain GV3101 and then into Arabidopsis plants by the floral dipping method²⁶.

Ovule clearance. To analyze seed and embryo phenotypes, siliques at different developmental stages after fertilization were dissected with a needle and quickly cleared in Herr's solution (lactic acid:chloral hydrate:phenol:clove oil:xylene = 2:2:2:2:1, wt/wt) as described previously²⁷. Images were acquired using an Olympus IX71 inverted microscope with differential interference contrast optics.

Confocal microscopy and image analysis. Ovules and early seeds were observed using a confocal microscope (Leica TCS SP8) and excited with a 488-nm laser and emitted light measured at 498–550 nm for enhanced GFP as well as excited with a 552-nm laser and emitted light measured at 600–650 nm for RFP. 3D images were generated using the LAS-X software (Leica) and 3D reconstructions were produced by Leica Aivia AI Image Analysis Software.

Flow cytometry. For flow cytometry analysis, two leaves were each harvested and chopped using a razor blade in a petri dish with 500 μl of nuclei extraction buffer (Partec CyStain). 1 ml of staining reagent (Partec CyStain UV Precise-Kit) was added and specimens were incubated at room temperature for 1 min. Samples were passed through a 40 µM nylon mesh into a 5 mL round-bottom polystyrene test tube (FALCON, 352235) and filtrates were analyzed using a Beckmann CytoFlex ploidy analyzer.

Chromosome spreads. For chromosome spreads, flower buds fixed in Carnoy’s solution were washed twice with 10 mM citrate buffer (pH 4.5) and digested with 0.3% cellulose and 0.3% pectolyase in citrate buffer at 25℃ for 1 h. After being washed twice with the same buffer and by adding DAPI solution (10μg/ml DAPI in citrate buffer), digested buds were squashed with a cover slip to release PMCs and samples were analyzed using a Leica SP8 CLSM.

Acknowledgements.

We thank Fred Berger for the HTR10–mRFP marker line. This work was supported by the National Natural Science Foundation of China (32130031; 31991201) and the China Postdoctoral Science Foundation (2021M702525, BX20200256, 2020M682466).

Author contributions

M.-X.S., X.Z., C.S. and T.D. designed the research plan. X.Z., C.S., S.L. and B.Z. performed the phenotype and genetics analyses. T.D. and M.-X.S. contributed to the data analysis and finalized the manuscript. All authors contributed to the data collection, presentation and manuscript writing.

Forster, B. P., Heberle-Bors, E., Kasha, K. J., & Touraev, A. The resurgence of haploids in higher plants. Trends Plant Sci. 12, 368–375 (2007).
Dunwell, J. M. Haploids in flowering plants: origins and exploitation. Plant Biotechnol J.8, 377–424 (2010).
Jacquier, N. M. et al. Puzzling out plant reproduction by haploid induction for innovations in plant breeding. Nat. Plants6, 610–619 (2020).
Gilles, L. M., Martinant, J. P., Rogowsky, P. M., & Widiez, T. Haploid induction in plants. Curr. Biol. 27, R1095–R1097 (2017).
Kalinowska, K., et al. State-of-the-art and novel developments of in vivo haploid technologies. Theor. Appl. Genet.132, 593–605 (2019).
Khanday, I., Skinner, D., Yang, B., Mercier, R., & Sundaresan, V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds. Nature 565, 91–95 (2019).
Conner, J.A., Podio, M., & Ozias-Akins, P. Haploid embryo production in rice and maize induced by PsASGR-BBML transgenes.Plant Reprod. 30, 41-52. (2017).
Underwood, C. J., et al. A PARTHENOGENESIS allele from apomictic dandelion can induce egg cell division without fertilization in lettuce. Nat. Genet.54, 84–93 (2022).
Kelliher, T. et al. MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction. Nature542, 105–109 (2017).
Liu, C. et al. A 4-bp insertion at ZmPLA1 encoding a putative phospholipase A generates haploid induction in maize. Mol. Plant10, 520–522 (2017).
Gilles, L. M. et al. Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maize. EMBO J.36, 707–717 (2017).
Li, Y. et al. Loss-of-function alleles of ZmPLD3 cause haploid induction in maize. Nat. Plants7, 1579–1588 (2021)
Yao, L. et al. OsMATL mutation induces haploid seed formation in indicarice. Nat. Plants4, 530–533 (2018).
Liu, C. et al. Extension of the in vivo haploid induction system from diploid maize to hexaploid wheat. Plant Biotechnol. J.18, 316–318 (2020).
Zhong, Y. et al. Mutation of ZmDMP enhances haploid induction in maize. Nat. Plants5, 575–580 (2019).
Zhong, Y. et al. A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis. Nat. Plants6, 466–472 (2020).
Ravi, M. & Chan, S. W. Haploid plants produced by centromere-mediated genome elimination. Nature464, 615-618 (2010).
Wang, N., Gent, J. I., & Dawe, R. K. Haploid induction by a maizecenh3 null mutant. Sci. Adv. 7, eabe2299 (2021).
Lv, J. et al. Generation of paternal haploids in wheat by genome editing of the centromeric histone CENH3. Nat. Biotechnol.38, 1397-1401 (2020).
Ishii, T., Karimi-Ashtiyani, R., & Houben, A. Haploidization via chromosome elimination: means and mechanisms. Annu. Rev. Plant Biol.67, 421-438 (2016).
Kasha, K. J. & Kao, K. N. High frequency haploid production in barley (Hordeum vulgare L.). Nature225, 874-876 (1970).
Yu, X. et al. Fertilized egg cells secrete endopeptidases to avoid polytubey. Nature592, 433-437 (2021).
Spielman, M. et al. TETRASPORE is required for male meiotic cytokinesis in Arabidopsis thaliana. Development124, 2645-2657 (1997).
Kurup, S. et al. Marking cell lineages in living tissues. Plant J.42, 444-453 (2005).
Aw, S. J., Hamamura, Y., Chen, Z., Schnittger, A., & Berger, F. Sperm entry is sufficient to trigger division of the central cell but the paternal genome is required for endosperm development in Arabidopsis. Development, 137, 2683-2690 (2010).
Clough, S. J., & Bent, A. F. Floral dip: a simplified method for Agrobacterium mediated transformation of Arabidopsis thaliana. Plant J.16, 735-743 (1998).
Ding, Y. H., Liu, N. Y., Tang, Z. S., Liu, J., & Yang, W. C. ArabidopsisGLUTAMINE-RICH PROTEIN23 is essential for early embryogenesis and encodes a novel nuclear PPR motif protein that interacts with RNA polymerase II subunit III. Plant Cell, 18, 815-830 (2006).

There is NO Competing Interest.

Supplementaryinformation.docx

PDF

Version 1

posted 15 Feb, 2022

You are reading this latest preprint version

A female in vivo haploid-induction system via mutagenesis of egg cell-specific peptidases

Status:

Version 1

Abstract

Figures

Full Text

Methods

Declarations

References

Competing Interests

Supplementary Files

Status:

Version 1