Obtaining Mutant Pollen for Phenotypic Analysis and Pollen Tube Dual Staining

Zhong, Sheng; Wang, Zhijuan; Liu, Qu

doi:10.1007/978-1-0716-0672-8_12

Cited by 3 publications

(3 citation statements)

References 24 publications

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“…To assess preferential fertilization, pollen grain separation and collection were performed as previously reported (Zhong et al, 2020). The cdka;1 mutant pollen grains (GFP–) were isolated from the complemented line ckda;1– / – carrying pCDKA;1:CDKA;1‐pLAT52:GFP+ /– under a BX51 epifluorescence microscope by an eyelash pen.…”

Section: Methodsmentioning

confidence: 99%

Egg cell‐secreted aspartic proteases ECS1/2 promote gamete attachment to prioritize the fertilization of egg cells over central cells in Arabidopsis

Jiang

Stührwohldt

Liu

et al. 2022

JIPB

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Double fertilization is an innovative phenomenon in angiosperms, in which one sperm cell first fuses with the egg cell to produce the embryo, and then the other sperm fuses with the central cell to produce the endosperm. However, the molecular mechanism of the preferential fertilization of egg cells is poorly understood. In this study, we report that two egg cell‐secreted aspartic proteases, ECS1 and ECS2, play an important role in promoting preferential fertilization of egg cells in Arabidopsis. We show that simultaneous loss of ECS1 and ECS2 function resulted in an approximately 20% reduction in fertility, which can be complemented by the full‐length ECS1/2 but not by corresponding active site mutants or by secretion‐defective versions of ECS1/2. Detailed phenotypic analysis revealed that the egg cell–sperm cell attachment was compromised in ecs1 ecs2 siliques. Limited pollination assays with cyclin‐dependent kinase a1 (cdka;1) pollen showed that preferential egg cell fertilization was impaired in the ecs1 ecs2 mutant. Taken together, these results demonstrate that egg cells secret two aspartic proteases, ECS1 and ECS2, to facilitate the attachment of sperm cells to egg cells so that preferential fertilization of egg cells is achieved. This study reveals the molecular mechanism of preferential fertilization in Arabidopsis thaliana.

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Section: Methodsmentioning

confidence: 99%

Egg cell‐secreted aspartic proteases ECS1/2 promote gamete attachment to prioritize the fertilization of egg cells over central cells in Arabidopsis

Jiang

Stührwohldt

Liu

et al. 2022

JIPB

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“…In the other strategy, gametophytes carrying the mutated gene are distinguished by fluorescence labels. This approach has been employed to successfully separate and collect mutant pollen of vacuolar protein sorting‐associated protein 41 (VPS41) and VPS18, two essential proteins involved in endomembrane trafficking (Hao et al, 2016; Zhong et al, 2020; Hou et al, 2021).…”

Section: Resultsmentioning

confidence: 99%

“…The complementary line was further transformed with the sperm nucleus marker pHTR10:HTR10‐RFP . To populate a collection of cdka;1 pollen, we manually picked out the ones with GFP− and red fluorescent protein (RFP)+ signals, as described in previous reports (Hao et al, 2016; Zhong et al, 2020). In contrast to the wild‐type pollen grain 98.8 ± 0.3% of which contains two sperm cells ( n = 335), 99.7 ± 0.5% of these cdka;1 pollen grains contained a single SLC ( n = 314), as indicated by the sperm cell reporter as well as 4ʹ,6‐diamidino‐2‐phenylindole (DAPI) staining (Figure 1C, D, F).…”

Section: Resultsmentioning

confidence: 99%

The egg cell is preferentially fertilized in Arabidopsis double fertilization

Hou

Wang

et al. 2022

JIPB

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In flowering plants (angiosperms), fertilization of the egg cell by one sperm cell produces an embryo, whereas fusion of a second sperm cell with the central cell generates the endosperm. In most angiosperms like Arabidopsis, a pollen grain contains two isomorphic sperm cells required for this double fertilization process. A long‐standing unsolved question is whether the two fertilization events have any preference. A tool to address this question is the usage of the cyclin‐dependent kinase a1 (cdka;1) mutant pollen, which produces a single sperm‐like cell (SLC). Here, we first adopt a complementation‐based fluorescence‐labeling method to successfully separate and collect cdka;1 mutant pollen containing a single SLC. Single‐cell RNA‐sequencing analysis revealed that cdka;1 SLCs show a gene expression profile highly similar to that of sperm cells and not to the generative cell, precursor of the two sperm cells. Pollination assays using a limited number of cdka;1 mutant pollen revealed that in 98.2% of the ovules, single fertilization of the egg cell occurred. Pollination of pistils with excessive cdka;1 mutant pollen allowed the delivery of a second SLC via fertilization recovery, which fertilized the central cell, resulting in 20.7% double‐fertilized ovules. This indicates that cdka;1 SLCs are able to fertilize both the egg and the central cell. Taken together, our findings have answered a long‐standing question and support that preferential fertilization of the egg cell is evident in Arabidopsis.

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VPS18-regulated vesicle trafficking controls the secretion of pectin and its modifying enzyme during pollen tube growth in Arabidopsis

et al. 2021

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In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33 and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells.

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Obtaining Mutant Pollen for Phenotypic Analysis and Pollen Tube Dual Staining

Cited by 3 publications

References 24 publications

Egg cell‐secreted aspartic proteases ECS1/2 promote gamete attachment to prioritize the fertilization of egg cells over central cells in Arabidopsis

Egg cell‐secreted aspartic proteases ECS1/2 promote gamete attachment to prioritize the fertilization of egg cells over central cells in Arabidopsis

The egg cell is preferentially fertilized in Arabidopsis double fertilization

VPS18-regulated vesicle trafficking controls the secretion of pectin and its modifying enzyme during pollen tube growth in Arabidopsis

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