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Expression of Brassica napus GLO1 is sufficient to breakdown artificial self-incompatibility in Arabidopsis thaliana

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Abstract

Members of the Brassicaceae family have the ability to regulate pollination events occurring on the stigma surface. In Brassica species, self-pollination leads to an allele-specific interaction between the pollen small cysteine-rich peptide ligand (SCR/SP11) and the stigmatic S-receptor kinase (SRK) that activates the E3 ubiquitin ligase ARC1 (Armadillo repeat-containing 1), resulting in proteasomal degradation of various compatibility factors including glyoxalase I (GLO1) which is necessary for successful pollination. In Brassica napus, the suppression of GLO1 was sufficient to reduce compatibility, and overexpression of GLO1 in self-incompatible Brassica napus stigmas resulted in partial breakdown of the self-incompatibility response. Here, we verified if BnGLO1 could function as a compatibility factor in the artificial self-incompatibility system of Arabidopsis thaliana expressing AlSCRb, AlSRKb and AlARC1 proteins from A. lyrata. Overexpression of BnGLO1 is sufficient to breakdown self-incompatibility response in A. thaliana stigmas. Therefore, GLO1 has an indisputable role as a compatibility factor in the stigma in regulating pollen attachment and pollen tube growth. Lastly, this study demonstrates the usefulness of an artificial self-incompatibility system in A. thaliana for interspecific self-incompatibility studies.

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Funding

Support for the Indriolo laboratory was provided by university start-up funds provided to Emily Indriolo from NMSU. Support for undergraduate researcher Patrick Kenney was provided by the Howard Hughes Medical Institute, Research Scholars Program: #52008103.

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  1. Patrick Kenney

    Present address: Division of Plant Sciences, University of Missouri, Waters Hall 1112 University Ave, Columbia, MO, 65201, USA

Authors and Affiliations

  1. Department of Biology, New Mexico State University, 1200 S. Horseshoe Dr, Las Cruces, NM, 88003, USA

    Patrick Kenney, Michael Balogh & Emily Indriolo

  2. Department of Botany and Plant Pathology, Purdue University, West Lafayette, 47907, IN, USA

    Subramanian Sankaranarayanan

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Contributions

SS and EI initiated the project and designed research; PK, SS, MB and EI performed all of the experiments. MB and EI statistically analyzed the results. EI and SS wrote the manuscript. EI obtained funding and supervised the project. All authors discussed the results and commented on the manuscript. The authors would lastly like to thank Ms. Alejandra Cobos and Dr. Marcus Samuel for critical feedback on the manuscript.

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Correspondence to Emily Indriolo.

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Communicated by Dolf Weijers.

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497_2020_392_MOESM1_ESM.tif

Supplemental Fig. 1. Additional Aniline Blue stains of A. thaliana stigmas 2 h post pollination. Manual self-pollination results of Sha wild-type compatible pollinations, Sha incompatible pollinations of AlARC1 + AlSRKb + AlSCRb lines and the Sha BnGLO1 + AlARC1 + AlSRKb + AlSCRb lines. Scale bar = 50 μm. (TIFF 8029 kb)

497_2020_392_MOESM2_ESM.eps

Supplemental Fig. 2. The reproductive expression data series of Arabidopsis thaliana Glyoxalase family members generated with (https://www.genevestigator.com). (EPS 2483 kb)

497_2020_392_MOESM3_ESM.eps

Supplemental Fig. 3. PCR genotyping of all transgenic A. thaliana lines. A. thaliana Sha plants were genotyped for the presence of transgenes. Line 61, 76 and 81 were the plants that were positive for the presence of AlARC1, AlSCRb-AlSRKb and BnGLO1 with primers that were specific for each of these constructs. Sizes for each PCR product are included in the figure. The previously characterized A. thaliana Sha artificial self-incompatible lines 1, 2 and 5 were confirmed by PCR with the AlARC1 and the AlSCR-SRK constructs. (EPS 3272 kb)

497_2020_392_MOESM4_ESM.eps

Supplemental Fig. 4. qPCR of transgenic A. thaliana lines. Relative level of expression of AlSRKb, AlARC1 and AtTUB4 in BnGLO1 + AlARC1 + AlSRKb + AlSCRb lines 61, 76 and 81. Each sample had three technical replicates, and the entire plate was run two times. The expression of each gene was set to a baseline of zero using a Sha wild-type cDNA, and the expression from each transgenic line was subtracted from the wild-type expression to determine if there was an equal or increased level of expression in the transgenic A. thaliana lines. (EPS 1991 kb)

497_2020_392_MOESM5_ESM.eps

Supplemental Fig. 5. Self-incompatible aniline blue stain phenotypes. Brightfield and Aniline Blue stains of stigmas. Sha self-pollinations of BnGLO1 + AlARC1 + AlSRKb + AlSCRb lines 51, 68, and 83 show the rejection of self-pollen grains 2 h post pollination. Scale bar = 50μm. (EPS 4550 kb)

Supplemental Fig. 6. R script. R script used to analyze seed set and pollen grain adherence. (DOCX 12 kb)

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Kenney, P., Sankaranarayanan, S., Balogh, M. et al. Expression of Brassica napus GLO1 is sufficient to breakdown artificial self-incompatibility in Arabidopsis thaliana. Plant Reprod 33, 159–171 (2020). https://doi.org/10.1007/s00497-020-00392-y

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