Advertisement

Step-by-step protocols for rice gamete isolation

  • Chenxin Li
  • Hengping Xu
  • Scott D. RussellEmail author
  • Venkatesan SundaresanEmail author
Methods Paper
Part of the following topical collections:
  1. Cellular Omics Methods in Plant Reproduction Research

Abstract

Key message

A detailed, step-by-step protocol for isolation of rice gametes for transcriptional profiling, with a general workflow that includes controls for RNA contamination from surrounding cells and tissues is presented.

Abstract

Characterization of the transcriptome and other -omics studies of flowering plant gametes are challenging as a consequence of the small sizes and relative inaccessibility of these cells. Collecting such poorly represented cells is also complicated by potential contamination from surrounding sporophytic, adjacent gametophytic tissues and difficulties in extracting high-quality intact cells. Here we present detailed, step-by-step procedures for collecting intact, unfixed rice (Oryza sativa) egg cells and sperm cells without enzymatic treatments. In addition, we also present a general workflow for assessing sample purity by RT-PCR, using primers specific for marker genes preferentially expressed in surrounding cells and tissues. These protocols should facilitate future studies of genome-scale characterization of gametes in this important model crop.

Keywords

Oryza sativa Egg cell Sperm cell Gametes Transcriptomes 

Notes

Acknowledgements

We thank Imtiyaz Khanday, Jonathan Gent, Sarah Anderson and Daniel Jones for helpful advice for optimizing the experimental methods. We thank Debra Skinner for assistance in artwork. This research was funded by the National Science Foundation (Award No. IOS-1547760) and the USDA Agricultural Experiment Station (Project No. CA-D-XXX-6973-H).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

497_2019_363_MOESM1_ESM.pdf (476 kb)
Fig. 1: Comparison of egg synergid morphology after isolation. Figure 2: Bioanalyzer traces of egg and sperm total RNA. Table 1: Primers sequences and amplicon lengths (PDF 475 kb)

References

  1. Abiko M, Furuta K, Yamauchi Y, Fujita C, Taoka M, Isobe T, Okamoto T (2013) Identification of proteins enriched in rice egg or sperm cells by single-cell proteomics. PLoS ONE.  https://doi.org/10.1371/journal.pone.0069578 Google Scholar
  2. Anderson SN, Johnson CS, Jones DS, Conrad LJ, Gou X, Russell SD, Sundaresan V (2013) Transcriptomes of isolated Oryza sativa gametes characterized by deep sequencing: evidence for distinct sex-dependent chromatin and epigenetic states before fertilization. Plant J 76(5):729–741.  https://doi.org/10.1111/tpj.12336 CrossRefGoogle Scholar
  3. Anderson SN, Johnson CS, Chesnut J, Jones DS, Khanday I, Woodhouse M, Li C, Conrad LJ, Russell SD, Sundaresan V (2017) The zygotic transition is initiated in unicellular plant zygotes with asymmetric activation of parental genomes. Dev Cell 43(3):349.e4–358.e4.  https://doi.org/10.1016/j.devcel.2017.10.005 CrossRefGoogle Scholar
  4. Cook M, Thilmony R (2012) The OsGEX2 gene promoter confers sperm cell expression in transgenic rice. Plant Mol Biol Rep 30(5):1138–1148.  https://doi.org/10.1007/s11105-012-0429-3 CrossRefGoogle Scholar
  5. Gou X, Wang S, Chen F (2011) Isolation and cytological observation of viable sperm cells of rice. Acta Botanica Sinica 41(6):669–671.  https://doi.org/10.1360/zd-2013-43-6-1064 Google Scholar
  6. Kawahara Y, de la Bastide M, Hamilton JP, Kanamori H, McCombie WR, Ouyang S, Schwartz DC, Tanaka T, Wu J, Zhou S, Childs KL, Davidson RM, Lin H, Quesada-Ocampo L, Vaillancourt B, Sakai H, Lee SS, Kim J, Numa H, Itoh T, Buell CR, Matsumoto T (2013) Improvement of the Oryza sativa nipponbare reference genome using next generation sequence and optical map data. Rice 6(1):3–10.  https://doi.org/10.1186/1939-8433-6-4 CrossRefGoogle Scholar
  7. Kyozuka J, Shimamoto K, Ag OR (2002) Ectopic expression of OsMADS3, a rice ortholog of AGAMOUS, caused a homeotic transformation of lodicules to stamens in transgenic rice plants. Plant Cell Physiol 43(1):130–135CrossRefGoogle Scholar
  8. Lu XC, Gong HQ, Huang ML, Bai SL, He YB, Mao X, Geng Z, Li SG, Wei L, Yuwen JS, Xu ZH, Bai SN (2006) Molecular analysis of early rice stamen development using organ-specific gene expression profiling. Plant Mol Biol 61(6):845–861.  https://doi.org/10.1007/s11103-006-0054-3 CrossRefGoogle Scholar
  9. Ohnishi T, Takanashi H, Mogi M, Takahashi H, Kikuchi S, Yano K, Okamoto T, Fujita M, Kurata N, Tsutsumi N (2011) Distinct gene expression profiles in egg and synergid cells of rice as revealed by cell type-specific microarrays. Plant Physiol 155(2):881–891.  https://doi.org/10.1104/pp.110.167502 CrossRefGoogle Scholar
  10. Okada T, Endo M, Singh MB, Bhalla PL (2005) Analysis of the histone H3 gene family in Arabidopsis and identification of the male-gamete-specific variant AtMGH3. Plant J 44(4):557–568.  https://doi.org/10.1111/j.1365-313X.2005.02554.x CrossRefGoogle Scholar
  11. Okamoto T (2017) Analysis of proteins enriched in rice gamete. In: Schmidt A (ed) Plant germline development: methods and protocols. Springer, New York, pp 251–263.  https://doi.org/10.1007/978-1-4939-7286-9_20 CrossRefGoogle Scholar
  12. Russell SD, Gou XP, Wong CE, Wang X, Yuan T, Wei XP, Bhalla PL, Singh MB (2012) Genomic profiling of rice sperm cell transcripts reveals conserved and distinct elements in the flowering plant male germ lineage. New Phytol 195(3):560–573.  https://doi.org/10.1111/j.1469-8137.2012.04199.x CrossRefGoogle Scholar
  13. Russell SD, Jones DS, Anderson S, Wang X, Sundaresan V, Gou X (2017) Isolation of rice sperm cells for transcriptional profiling. In: Schmidt A (ed) Plant germline development: methods and protocols. Springer, New York, pp 211–219.  https://doi.org/10.1007/978-1-4939-7286-9_17 CrossRefGoogle Scholar
  14. Schon MA, Nodine MD (2017) Widespread contamination of Arabidopsis embryo and endosperm transcriptome data sets. Plant Cell 29(4):608–617.  https://doi.org/10.1105/tpc.16.00845 CrossRefGoogle Scholar
  15. Uchiumi T, Uemura I, Okamoto T (2007) Establishment of an in vitro fertilization system in rice (Oryza sativa L.). Planta 226(3):581–589.  https://doi.org/10.1007/s00425-007-0506-2 CrossRefGoogle Scholar
  16. Xiao H, Wang Y, Liu D, Wang W, Li X, Zhao X, Xu J, Zhai W, Zhu L (2003) Functional analysis of the rice AP3 homologue OsMADS16 by RNA interference. Plant Mol Biol 52(5):957–966.  https://doi.org/10.1023/A:1025401611354 CrossRefGoogle Scholar
  17. Xu HP, Tsao TH (1997) Detection and immunolocalization of glycoproteins of the plasma membrane of maize sperm cells. Protoplasma 198(3–4):125–129.  https://doi.org/10.1007/BF01287560 CrossRefGoogle Scholar
  18. Xu H, Weterings K, Vriezen W, Feron R, Xue Y, Derksen J, Mariani C (2002) Isolation and characterization of male-germ-cell transcripts in Nicotiana tabacum. Sex Plant Reprod 14(6):339–346.  https://doi.org/10.1007/s00497-002-0128-6 CrossRefGoogle Scholar
  19. Zhang J, Dong WH, Galli A, Potrykus I (1999) Regeneration of fertile plants from isolated zygotes of rice (Oryza sativa). Plant Cell Rep 19(2):128–132.  https://doi.org/10.1007/s002990050722 CrossRefGoogle Scholar
  20. Zhang YN, Wei DM, He EM, Miao S, Tian HQ, Russell SD (2010) Isolation of male and female gametes of rice. Crop Sci 50(6):2457-2463.  https://doi.org/10.2135/cropsci2010.02.0066 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Plant BiologyUniversity of CaliforniaDavisUSA
  2. 2.Department of Microbiology and Plant BiologyUniversity of OklahomaNormanUSA
  3. 3.Department of Plant SciencesUniversity of CaliforniaDavisUSA

Personalised recommendations