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Sporophyte-directed gametogenesis in Arabidopsis

Nature Plants volume 11, pages 398–409 (2025)Cite this article

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Abstract

Plants alternate between diploid sporophyte and haploid gametophyte generations1. In mosses, which retain features of ancestral land plants, the gametophyte is dominant and has an independent existence. However, in flowering plants the gametophyte has undergone evolutionary reduction to just a few cells enclosed within the sporophyte. The gametophyte is thought to retain genetic control of its development even after reduction2. Here we show that male gametophyte development in Arabidopsis, long considered to be autonomous, is also under genetic control of the sporophyte via a repressive mechanism that includes large-scale regulation of protein turnover. We identify an Arabidopsis gene SHUKR as an inhibitor of male gametic gene expression. SHUKR is unrelated to proteins of known function and acts sporophytically in meiosis to control gametophyte development by negatively regulating expression of a large set of genes specific to postmeiotic gametogenesis. This control emerged late in evolution as SHUKR homologues are found only in eudicots. We show that SHUKR is rapidly evolving under positive selection, suggesting that variation in control of protein turnover during male gametogenesis has played an important role in evolution within eudicots.

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Fig. 1: SHUKR is required for pollen development.
Fig. 2: Staging of skr phenotype and SKR protein expression.
Fig. 3: SHUKR inhibits expression of gametogenesis genes.
Fig. 4: SHUKR regulates the proteasome in pollen development.
Fig. 5: Evolutionary analysis of the SHUKR domain and evidence for positive selection.

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Data availability

All data in support of the results are included in the article and in Supplementary Information. Sequencing data are deposited in the NCBI Sequence Read Archive, BioProject identifier PRJNA1164421. The TAIR10 reference genome was taken from The Arabidopsis Information Resource database at https://www.arabidopsis.org/download/list?dir=Sequences%2FTAIR10_blastsets, and the Ler sequence was downloaded from https://1001genomes.org/projects/MPIPZJiao2020/index.html. The following additional databases were used: https://cgpdb.ucdavis.edu/cgpdb2/data_analysis/reference_genome_set/Arabidopsis/A_thaliana_ATGC_2006_08_24.protein.COS_STRICT.IDs.txt for single-copy Arabidopsis genes, and CoNekT at https://evorepro.sbs.ntu.edu.sg. Correspondence and requests for materials should be addressed to the corresponding author. Source data are provided with this paper.

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Acknowledgements

We thank D. Twell for kindly providing the LAT52–GFP reporter line, ABRC and CSHL for seeds, K. Bannerjee and M. Kamble for technical support, M. Sai Kiran for maintenance of plants, the NGS and Advanced Microscopy facilities at CCMB and P. Pant for help with illustrations. We thank E. Meyerowitz, U. Nath and M. Reddy for valuable comments on the article. This work was supported by a CSIR Network Project grant MLP0120 and a Department of Biotechnology Centre of Excellence grant BT/PR21305/COE/34/44/2016 to I.S. S.P. and P.S. were supported by CSIR Fellowships. A.R. was supported by a Department of Biotechnology Research Associate Fellowship. I.S. acknowledges a JC Bose fellowship from the Department of Science and Technology and a senior scientist fellowship from the Indian National Science Academy.

Author information

Author notes

  1. Saurabh Pandey

    Present address: databaum GmbH, Hamburg, Germany

  2. A. Ramesha

    Present address: Seri-Biotech Research Laboratory, Central Silk Board, Bangalore, India

  3. Aparna Singh

    Present address: Department of Botany, MMV, Banaras Hindu University, Varanasi, India

  4. Chandan Kumar

    Present address: University of Texas at Austin, Austin, TX, USA

  5. Deceased: Veeraputhiran Subbiah.

  6. These authors contributed equally: Prakash Sivakumar, Saurabh Pandey, A. Ramesha.

Authors and Affiliations

  1. Centre for Cellular and Molecular Biology, CSIR, Hyderabad, India

    Prakash Sivakumar, Saurabh Pandey, A. Ramesha, Jayeshkumar Narsibhai Davda, Aparna Singh, Chandan Kumar, Hardik Gala, Veeraputhiran Subbiah, Harikrishna Adicherla, Jyotsna Dhawan & Imran Siddiqi

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India

    Prakash Sivakumar & Imran Siddiqi

  3. National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA

    L. Aravind

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  1. Prakash Sivakumar
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Contributions

P.S., S.P., J.N.D., J.D. and I.S. conceived and planned experiments. S.P., P.S., A.R., J.N.D., V.S., H.A., A.S. and I.S. performed experiments. S.P., P.S., A.R., A.S., C.K., H.G., J.D. and I.S. provided analysis of the experimental results. L.A. contributed the evolutionary analysis of Fig. 5. I.S. and P.S. wrote the manuscript, with contributions from L.A. and J.D.

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Correspondence to Imran Siddiqi.

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Extended data

Extended Data Fig. 1 skr mutant alleles show male sterility.

(a) Gene diagram of SKR showing location of insertions: skr-1 (GT_5_101780), skr-2 (CSHL_GT5061), skr-3 (SALKSeq_034497.1). (b) Toluidine blue stained tetrads. The experiment was repeated twice. (c) Inflorescence stem and Alexander staining showing recessive male sterility of skr-1. Mean and S.D. of viable pollen counts per anther (7–12 anthers from each of 4 plants) for WT = 400 +/−51; skr-1/+ = 395 +/−39; skr-1 = 0.9+/−2.1 (d) Complementation of skr-3 by a gSKR transgene. Scale bar: (b) 20 µm; (c, d) 1 cm for inflorescence, 100 µm for anthers.

Extended Data Fig. 2 Profiling of SKR expression in male germline development.

TPM values for SKR were collected from the CoNekt database, except for meiocytes. Meiocyte TPM value were taken from (Walker61). UNM – Uninucleate microspore; BCP – Bicellular pollen; TCP – Tricellular pollen; MP – Mature pollen.

Extended Data Fig. 3 Sections of anther development stages.

Corresponding stages are shown for wild type (A-D, I-L) and skr-1 (E-H, M-P). Stages are numbered according to (Sanders49). Abbreviations: E epidermis, En endothecium, ML middle layer, T tapetum, Td tetrad, Msp microspore, DM degenerating microspore, PG pollen grain, St stomium, MD microspore debris. Scale bar 20 µm. The experiment was repeated repeated twice.

Extended Data Fig. 4 Staging of microspore degeneration in skr-1.

Microspore stage and phenotypes are shown together with stage of ovules from the same bud (Schneitz50) as a reference for comparison to wild type. Scale bar: 10 µm. The experiment was performed on inflorescences from three plants for each genotype.

Extended Data Fig. 5 Ultrastructure and marker expression reveal early gametogenesis defects in skr.

(a–c) Ultrastructure of tetrad and early uninucleate microspores showing onset of mutant phenotype at tetrad stage in skr-1. (a) WT: Normal tetrad and microspore; skr-1: normal tetrad, degenerating microspore with retracted PM. (b) skr-1 tetrad in which one spore shows PM retraction from callose wall. (c) Magnified image of boxed region from (b) showing PM retraction from callose wall (Cal) and disrupted cell wall material (arrowhead). For 3a-c, buds from at least three plants were examined for each genotype and showed similar results. (d) Quantitation of normal spores and spores showing PM retraction and broken cell wall material (R&B) in tetrads (WT: 104 normal, 1 abnormal; skr-1: 102 normal, 25 abnormal; two-sided Fisher’s exact test p = 1.33e-06). (e) Presence of two LAT52:GFP signals in a proportion of skr-1 microspores (WT: 0/58; skr-1: 10/28; two-sided Fisher’s exact test p = 4.19e-05). Scale bar (a): Tetrad images 5 µm; microspore images 2 µm; (b) 5 µm; (c) 1 µm; (e) 10 µm.

Extended Data Fig. 6 Precocious expression of F-box gene:nlsGFP fusions in meiosis in skr-1.

(a) At5g02980. A-C, A’-C’: wild type; D-F, D’-F’: skr-1; A,A’,D,D’ meiocytes; B,B’,E,E’: tetrad; C,C’,F,F’: uninucleate microspores. (b) At5g62510. A-E, A’-E’: wild type; F-H, F’-H’: skr-1; A,A’,F,F’ meiocytes; B,B’,G,G’: tetrad; C-E, C’-E’,H,H’: uninucleate microspores. A-H: GFP fluorescence; A’-H’: corresponding DIC. GFP expression in skr-1 is first detected in meiocytes (At5g02980) or tetrad stage At5g62510) whereas in wild type earliest GFP signal was in released microspores. The experiment was repeated using three independent mutant and wild type plants for each line. Scale bar: 5 μm.

Extended Data Fig. 7 ski2 is a suppressor of skr.

(a) Mendelian segregation of the suppressor mutation in the 08-10 skr-1 suppressor line (b) A C > T transition in the ski2 allele (named ski2-7) present in the 08-10 skr-1 suppressor line results in a TGA termination codon at position 518. (c) Restoration of fertility and pollen viability in skr-3 by the independent allele ski2-6. Scale bar: inflorescence 1 cm; anthers 100 μm.

Extended Data Fig. 8 Phylogenetic tree of the SHUKR family from eudicots.

119 representatives from 85 species were used for tree construction using only the SHUKR domain that is shared by all representatives of the family. Genbank ID and species are indicated. Brassicaceae+Cleomaceae (dark green) and Asteraceae (light green).

Extended Data Fig. 9 Phylogenetic tree of Brassicales SHUKR and SHUKR-like (SKL) genes used for branch-site test.

The SHUKR gene family is represented by a single gene in Carica papaya, whereas Brassicaceae and Cleomaceae experienced a gene duplication event prior to their divergence. CDS were aligned using MAFFT, and RAxML was used to build the phylogenetic tree with 1000 bootstraps. The red branch was selected as foreground for SKR. Branch-site test for positive selection was performed using the CODEML package implemented in EASYCoDEML. Likelihood Ratio Test (LRT) results of Model A vs Model A null (Extended Data Table 1), gave a p-value of 0.0031 for SKR. The tree was unrooted.

Extended Data Table 1 Branch-site test results for positive selection in the SKR branch in the Brassicaceae
Full size table

Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and File 1.

Reporting Summary

Supplementary Table 1

Differentially expressed gene lists.

Supplementary Table 2

Male gametogenesis enrichment analysis of skr-1 upregulated genes.

Supplementary Table 3

Analysis of genes differentially expressed in skr-1.

Supplementary Table 4

Differentially expressed translation-related genes in skr-1 ski2.

Supplementary Table 5

Enrichment for short taxonomic scale (STS) F-box genes upregulated in skr-1.

Supplementary Table 6

List of primers.

Source data

Source Data Fig. 1

Uncropped Fig. 1c and unprocessed Fig. 1d.

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Sivakumar, P., Pandey, S., Ramesha, A. et al. Sporophyte-directed gametogenesis in Arabidopsis. Nat. Plants 11, 398–409 (2025). https://doi.org/10.1038/s41477-025-01932-y

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