Abstract
During sexual reproduction in flowering plants, pollen grains germinate on the stigma surface and grow through the stigma-style tissue to reach the ovary and deliver sperm cells for fertilization. Here, we outline a method to test whether a pollen fertility mutation specifically disrupts pollen penetration through the stigma-style barrier. This method surgically removes the stigma-style (stigma decapitation) to test whether transferring pollen directly onto an exposed ovary surface significantly improves the transmission efficiency (TE) of a mutant allele. To illustrate this technique, we applied stigma decapitation to investigate a loss-of-function mutation in Arabidopsis OFT1, a gene encoding a putative o-fucosyl transferase functioning in the secretory pathway. oft1-3 mutant pollen showed a significant decrease in transmission efficiency compared to wild type. Decapitation crosses (described here) indicated that the removal of the stigma-style barrier alleviated the transmission deficiency from 858-fold to a 2.6-fold, providing evidence that most, but not all, oft1 pollen deficiencies can be attributed to a reduced ability to penetrate through the stigma-style barrier. This method outlines a genetic strategy to quantify a mutation’s impact on the ability of pollen to navigate through the stigma-style barrier on its journey to the ovule.
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References
Johnson MA, Harper JF, Palanivelu R (2019) A fruitful journey: pollen tube navigation from germination to fertilization. Annu Rev Plant Biol 70:809–837. https://doi.org/10.1146/annurev-arplant-050718-100133
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Supplemental Table 1
Excel spreadsheet template for calculating transmission efficiencies (TE), TE ratio (TEr), and p-values from a chi-square test. (A) Generic worksheet for genetic calculations after filling in number of progeny that are resistant (#R) and sensitive (#S) for the selection marker being scored (purple columns). Observed TE and TEr are shown in yellow, and p-value is shown in green column. Only enter #R and #S on lines #1–6 because “b” crosses will automatically prefill from these entries. Caution: This sheet contains many embedded formulas. We recommend saving an unaltered version for future use. (B) Worksheet with actual transmission frequencies that support a model in which pollen tube penetration requires an OFT1 (results from Smith et al., https://doi.org/10.1104/pp.17.01577). Note that expected TEs can vary depending on the hypothesis being tested. TG = transgene. (XLSX 26 kb)
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Weigand, C., Harper, J. (2020). Decapitation Crosses to Test Pollen Fertility Mutations for Defects in Stigma-Style Penetration. In: Geitmann, A. (eds) Pollen and Pollen Tube Biology. Methods in Molecular Biology, vol 2160. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0672-8_3
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DOI: https://doi.org/10.1007/978-1-0716-0672-8_3
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