Skip to main content
SpringerLink
Log in

Functional Characterization of OsRac6 Involved in Pollen Germination in Rice

  • Research Article
  • Published:
Journal of Plant Biology Aims and scope Submit manuscript

Abstract

For a successful reproduction in angiosperms, pollen germination and pollen tube growth must proceed normally. The processes are regulated by transcriptional regulation and cellular activities, in which signaling proteins are the key. Ras homolog (Rho)-type GTPases (Rops), which are known as RACs, are a group of Rho small GTP-binding proteins which evolved in plants. However, the molecular mechanisms of the rice Rac family (OsRac) genes in pollen germination are not yet understood. In this investigation, the OsRac6 gene, which is only expressed only during the pollen development stage, was identified. A functional characterization of OsRac6 using gene-edited mutants exhibits male sterility due to the disruption of pollen germination. Then, to determine downstream candidate genes modulated by the OsRac6 genes during pollen development, we performed RNA-seq analysis by comparing osrac6-1 and wild-type (WT) anthers. Subsequently, we identified 652 genes with pollen-preferential expression which were upregulated compared with the WT, and 608 genes which were downregulated. Furthermore, gene ontology (GO) enrichment and MapMan analyses show that downregulated genes are closely associated with transcription, protein ubiquitination, proteolysis, cell wall modification, cell organization, cell cycle, and calcium regulation. The results of this study will help in elucidating the molecular mechanisms of pollen germination in rice mediated by OsRac6.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Data availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

References

  • Boonburapong B, Buaboocha T (2007) Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins. BMC Plant Biol 7:4

    Article  PubMed  PubMed Central  Google Scholar 

  • Chandran AKN, Hong W-J, Abhijith B, Lee J, Kim Y-J, Park SK, Jung K-H (2020) Rice male gamete expression database (RMEDB): a web resource for functional genomic studies of rice male organ development. J Plant Biol 63:421–430

    Article  Google Scholar 

  • Christensen TM, Vejlupkova Z, Sharma YK, Arthur KM, Spatafora JW, Albright CA, Meeley RB, Duvick JP, Quatrano RS, Fowler JE (2003) Conserved subgroups and developmental regulation in the monocot rop gene family. Plant Physiol 133:1791–1808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Craddock C, Lavagi I, Yang Z (2012) New insights into rho signaling from plant ROP/Rac GTPases. Trends Cell Biol 22:492–501

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dodou E, Treisman R (1997) The Saccharomyces cerevisiae MADS-box transcription factor Rlm1 is a target for the Mpk1 mitogen-activated protein kinase pathway. Mol Cell Biol 17:1848–1859

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dorjgotov D, Jurca ME, Fodor-Dunai C, Szűcs A, Ötvös K, Klement É, Bíró J, Fehér A (2009) Plant Rho-type (Rop) GTPase-dependent activation of receptor-like cytoplasmic kinases in vitro. FEBS Lett 583:1175–1182

    Article  CAS  PubMed  Google Scholar 

  • Feng H, Liu C, Fu R, Zhang M, Li H, Shen L, Wei Q, Sun X, Xu L, Ni B, Li C (2019) Lorelei-like GPI-anchored proteins 2/3 regulate pollen tube growth as chaperones and coreceptors for ANXUR/BUPS receptor kinases in Arabidopsis. Mol Plant 12:1612–1623

    Article  CAS  PubMed  Google Scholar 

  • Gibieža P, Petrikaitė V (2021) The regulation of actin dynamics during cell division and malignancy. Am J Cancer Res 11:4050–4069

    PubMed  PubMed Central  Google Scholar 

  • Hoefle C, McCollum C, Hückelhoven R (2020) Barley ROP-Interactive Partner-a organizes into RAC1- and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1-dependent membrane domains. BMC Plant Biol 20:94

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hoffmann RD, Portes MT, Olsen LI, Damineli DSC, Hayashi M, Nunes CO, Pedersen JT, Lima PT, Campos C, Feijó JA, Palmgren M (2020) Plasma membrane H+-ATPases sustain pollen tube growth and fertilization. Nat Commun 11:2395

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hong W-J, Kim Y-J, Chandran AKN, Jung K-H (2019) Infrastructures of systems biology that facilitate functional genomic study in rice. Rice 12:15

    Article  PubMed  PubMed Central  Google Scholar 

  • Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun 345:646–651

    Article  CAS  PubMed  Google Scholar 

  • Jeon JS, Lee S, Jung KH, Jun SH, Jeong DH, Lee J, Kim C, Jang S, Yang K, Nam J, An K, Han MJ, Sung RJ, Choi HS, Yu JH, Choi JH, Cho SY, Cha SS, Kim SI, An G (2000) T-DNA insertional mutagenesis for functional genomics in rice. Plant J 22:561–570

    Article  CAS  PubMed  Google Scholar 

  • Jiang M, Zhao C, Zhao M, Li Y, Wen G (2020) Phylogeny and evolution of calcineurin B-Like (CBL) gene family in grass and functional analyses of rice CBLs. J Plant Biol 63:117–130

    Article  CAS  Google Scholar 

  • Kim Y-J, Jung K-H (2023) WD40-domain protein GORI is an integrative scaffold that is required for pollen tube growth in rice. Plant Signal Behav 18:2082678

    Article  PubMed  Google Scholar 

  • Kim D, Paggi J-M, Park C, Bennett C, Salzberg S-L (2019) Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotech 37:907–915

    Article  CAS  Google Scholar 

  • Kim E-J, Park S-W, Hong W-J, Silva J, Liang W, Zhang D, Jung K-H, Kim Y-J (2020) Genome-wide analysis of RopGEF gene family to identify genes contributing to pollen tube growth in rice (Oryza sativa). BMC Plant Biol 20:95

    Article  PubMed  PubMed Central  Google Scholar 

  • Kim J, Lee SB, Suh MC (2021a) Arabidopsis 3-Ketoacyl-CoA synthase 4 is essential for root and pollen tube growth. J Plant Biol 64:155–165

    Article  CAS  Google Scholar 

  • Kim YJ, Kim MH, Hong WJ, Moon S, Kim EJ, Silva J, Lee J, Lee S, Kim ST, Park SK, Jung KH (2021b) GORI, encoding the WD40 domain protein, is required for pollen tube germination and elongation in rice. Plant J 105:1645–1664

    Article  CAS  PubMed  Google Scholar 

  • Klahre U, Kost B (2006) Tobacco RhoGTPase activating protein1 spatially restricts signaling of RAC/Rop to the apex of pollen tubes. Plant Cell 18:3033–3046

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee SK, Hong WJ, Silva J, Kim EJ, Park SK, Jung KH, Kim YJ (2021) Global identification of ANTH genes involved in rice pollen germination and functional characterization of a key member, OsANTH3. Front Plant Sci. https://doi.org/10.3389/fpls.2021.609473

    Article  PubMed  PubMed Central  Google Scholar 

  • Li H, Lin Y, Heath R, Zhu M, Yang Z (1999) Control of pollen tube tip growth by a rop GTPase-dependent pathway that leads to tip-localized calcium influx. Plant Cell 11:1731–1742

    CAS  PubMed  PubMed Central  Google Scholar 

  • Li H, Luo N, Wang W, Liu Z, Chen J, Zhao L, Tan L, Wang C, Qin Y, Li C, Xu T, Yang Z (2018) The REN4 rheostat dynamically coordinates the apical and lateral domains of Arabidopsis pollen tubes. Nat Commun 9:2573

    Article  PubMed  PubMed Central  Google Scholar 

  • Liao Y, Smyth GK, Shi W (2014) Featurecounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30:923–930

    Article  CAS  PubMed  Google Scholar 

  • Liu L, Zheng C, Kuang B, Wei L, Yan L, Wang T (2016) Receptor-like kinase RUPO interacts with potassium transporters to regulate pollen tube growth and integrity in rice. PLoS Genet 12:e1006085

    Article  PubMed  PubMed Central  Google Scholar 

  • Love M-I, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550

    Article  PubMed  PubMed Central  Google Scholar 

  • Lu Y, Chanroj S, Zulkifli L, Johnson MA, Uozumi N, Cheung A, Sze H (2011) Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23:81–93

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Luo N, Yan A, Liu G, Guo J, Rong D, Kanaoka MM, Xiao Z, Xu G, Higashiyama T, Cui X, Yang Z (2017) Exocytosis-coordinated mechanisms for tip growth underlie pollen tube growth guidance. Nat Commun 8:1687

    Article  PubMed  PubMed Central  Google Scholar 

  • Mecchia MA, Santos-Fernandez G, Duss NN, Somoza SC, Boisson-Dernier A, Gagliardini V, Martínez-Bernardini A, Fabrice TN, Ringli C, Muschietti JP, Grossniklaus U (2017) RALF4/19 peptides interact with LRX proteins to control pollen tube growth in Arabidopsis. Science 358:1600–1603

    Article  CAS  PubMed  Google Scholar 

  • Molendijk AJ, Ruperti B, Singh MK, Dovzhenko A, Ditengou FA, Milia M, Westphal L, Rosahl S, Soellick T-R, Uhrig J, Weingarten L, Huber M, Palme K (2008) A cysteine-rich receptor-like kinase NCRK and a pathogen-induced protein kinase RBK1 are Rop GTPase interactors. Plant J 53:909–923

    Article  CAS  PubMed  Google Scholar 

  • Moon S, Oo MM, Kim B, Koh HJ, Oh SA, Yi G, An G, Park SK, Jung KH (2018) Genome-wide analyses of late pollen-preferred genes conserved in various rice cultivars and functional identification of a gene involved in the key processes of late pollen development. Rice (n Y) 11:28

    Article  PubMed  Google Scholar 

  • Nagawa S, Xu T, Yang Z (2010) RHO GTPase in Plants. Small GTPases 1:78–88

    Article  PubMed  PubMed Central  Google Scholar 

  • Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31:1120–1123

    Article  CAS  PubMed  Google Scholar 

  • Nakashima A, Chen L, Thao NP, Fujiwara M, Wong HL, Kuwano M, Umemura K, Shirasu K, Kawasaki T, Shimamoto K (2008) RACK1 functions in rice innate immunity by interacting with the Rac1 immune complex. Plant Cell 20:2265–2279

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Plotnikov A, Zehorai E, Procaccia S, Seger R (2011) The MAPK cascades Signaling components, nuclear roles and mechanisms of nuclear translocation. Biochim Biophys Acta (BBA) Mol Cell Res 1813:1619–1633

    Article  CAS  Google Scholar 

  • Schaefer A, Höhner K, Berken A, Wittinghofer A (2011) The unique plant RhoGAPs are dimeric and contain a CRIB motif required for affinity and specificity towards cognate small G proteins. Biopolymers 95:420–433

    Article  CAS  PubMed  Google Scholar 

  • Schappi JM, Krbanjevic A, Rasenick MM (2014) Tubulin, actin and heterotrimeric G proteins: Coordination of signaling and structure. Biochim Biophys Acta (BBA) Biomembr 1838:674–681

    Article  CAS  Google Scholar 

  • Shin NH, Trang DT, Hong WJ, Kang K, Chuluuntsetseg J, Moon JK, Yoo YH, Jung KH, Yoo SC (2020) Rice senescence-induced receptor-like kinase (OsSRLK) is involved in phytohormone-mediated chlorophyll degradation. Int J Mol Sci 21:260

    Article  CAS  Google Scholar 

  • Silver N, Best S, Jiang J, Thein SL (2006) Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 7:33

    Article  PubMed  PubMed Central  Google Scholar 

  • Smoot ME, Ono K, Ruscheinski J, Wang P-L, Ideker T (2011) Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics 27:431–432

    Article  CAS  PubMed  Google Scholar 

  • Tanaka I, Wakabayashi T (1992) Organization of the actin and microtubule cytoskeleton preceding pollen germination. Planta 186:473–482

    Article  CAS  PubMed  Google Scholar 

  • Tibshirani R, Walther G, Hastie T (2001) Estimating the number of clusters in a data set via the gap statistic. J Royal Stat Soc Ser B (stat Methodol) 63:411–423

    Article  Google Scholar 

  • Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562–578

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Usadel B, Poree F, Nagel A, Lohse M, Czedik-Eysenberg A, Stitt M (2009) A guide to using MapMan to visualize and compare omics data in plants: a case study in the crop species, Maize. Plant Cell Environ 32:1211–1229

    Article  PubMed  Google Scholar 

  • Vo KTX, Kim CY, Chandran AKN, Jung KH, An G, Jeon JS (2015) Molecular insights into the function of ankyrin proteins in plants. J Plant Biol 58:271–284

    Article  CAS  Google Scholar 

  • Vogler H, Santos-Fernandez G, Mecchia MA, Grossniklaus U (2019) To preserve or to destroy, that is the question: the role of the cell wall integrity pathway in pollen tube growth. Curr Opin Plant Biol 52:131–139

    Article  CAS  PubMed  Google Scholar 

  • Wamaitha MJ, Yamamoto R, Wong HL, Kawasaki T, Kawano Y, Shimamoto K (2012) OsRap2.6 transcription factor contributes to rice innate immunity through its interaction with receptor for activated kinase-C 1 (RACK1). Rice 5:35

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang HJ, Huang JC, Jauh GY (2010) Pollen Germination and Tube Growth. In: Advances in Botanical Research. pp 1–52

  • Xu Y, Cai W, Chen X, Chen M, Liang W (2022) A small Rho GTPase OsRacB is required for pollen germination in rice. Dev Growth Differ 64:88–97

    Article  CAS  PubMed  Google Scholar 

  • Yoon J, Cho LH, Jung KH (2022) Hierarchical structures and dissected functions of MADS-box transcription factors in rice development. J Plant Biol 65:99–109

    Article  CAS  Google Scholar 

  • Zhang D, Wilson ZA (2009) Stamen specification and anther development in rice. Chin Sci Bull 54:2342–2353

    Article  CAS  Google Scholar 

  • Zhao Z, Manser E (2005) PAK and other rho-associated kinases - effectors with surprisingly diverse mechanisms of regulation. Biochem J 386:201–214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhao T, Piñeyro-Nelson A, Yu Q, Pan X, Hu X, Liu H, Liao J (2021) Transcriptome-wide analyses provide insights into development of the hedychium coronarium flower, revealing potential roles of PTL. J Plant Biol 64:431–445

    Article  CAS  Google Scholar 

  • Zhou L, Lan W, Jiang Y, Fang W, Luan S (2014) A calcium-dependent protein kinase interacts with and activates a calcium channel to regulate pollen tube growth. Mol Plant 7:369–376

    Article  CAS  PubMed  Google Scholar 

  • Zhu L, Chu LC, Liang Y, Zhang XQ, Chen LQ, Ye D (2018) The Arabidopsis CrRLK1L protein kinases BUPS1 and BUPS2 are required for normal growth of pollen tubes in the pistil. Plant J 95:474–486

    Article  CAS  PubMed  Google Scholar 

  • Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) Genevestigator. Arabidopsis microarray database and analysis toolbox. Plant Physiol 136:2621–2632

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (NRF-2021R1A5A1032428 to K.-H.J.; NRF-2021R1A2C2010124 to S.-K.P.), a grant from the New Breeding Technologies Development Program (PJ01661002 to K.-H.J.) of the Rural Development Administration, Republic of Korea, and a grant from Kyungpook National University Research Fund, 2022 (S.-K.P.).

Author information

Authors and Affiliations

  1. Graduate School of Green-Bio Science and Crop Biotech Institute, Kyung Hee University, Yongin, 17104, South Korea

    Su Kyoung Lee, Eui-Jung Kim, Sunok Moon & Ki-Hong Jung

  2. Research Center for Plant Plasticity, Seoul National University, Seoul, 08826, Republic of Korea

    Su Kyoung Lee, Eui-Jung Kim, Sunok Moon & Ki-Hong Jung

  3. Department of Life Science and Environmental Biochemistry, Life and Industry Convergence Research Institute, Pusan National University, Miryang, 50463, South Korea

    Yu-Jin Kim

  4. School of Applied Bio Sciences, Kyungpook National University, Daegu, 41566, Republic of Korea

    Soon-Ki Park

  5. Department of Smart Farm Science, Kyung Hee University, Yongin, 17104, Republic of Korea

    Woo-Jong Hong

Authors
  1. Su Kyoung Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Woo-Jong Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eui-Jung Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sunok Moon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yu-Jin Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Soon-Ki Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ki-Hong Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

KHJ and SKP designed the research project. SKL, WJH, EJK, SM, and YJK performed the experiments. SKL and KHJ analyzed data. SKL, SKP, and KHJ wrote the manuscript.

Corresponding authors

Correspondence to Soon-Ki Park or Ki-Hong Jung.

Ethics declarations

Conflict of Interest

All authors declare that they have no conflicts of interest.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 1910 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lee, S.K., Hong, WJ., Kim, EJ. et al. Functional Characterization of OsRac6 Involved in Pollen Germination in Rice. J. Plant Biol. 66, 425–437 (2023). https://doi.org/10.1007/s12374-023-09403-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12374-023-09403-7

Keywords

Search

Navigation