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Fluorescence-tagged transgenic lines reveal genetic defects in pollen growth--application to the eIF3 complex.

Roy B, Copenhaver GP, von Arnim AG - PLoS ONE (2011)

Bottom Line: We also detected reduced pollen germination for eif3e.However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation. eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs.Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee, United States of America.

ABSTRACT

Background: Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3) cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. To accomplish this we have developed a broadly applicable method to track mutant alleles that are not already tagged by a visible marker gene through the male lineage of Arabidopsis.

Methodology/principal findings: Fluorescence tagged lines (FTLs) harbor a transgenic fluorescent protein gene (XFP) expressed by the pollen-specific LAT52 promoter at a defined chromosomal position. In the existing collection of FTLs there are enough XFP marker genes to track nearly every nuclear gene by virtue of its genetic linkage to a transgenic marker gene. Using FTLs in a quartet mutant, which yields mature pollen tetrads, we determined that the pollen transmission defect of the eif3h-1 allele is due to a combination of reduced pollen germination and reduced pollen tube elongation. We also detected reduced pollen germination for eif3e. However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation.

Conclusion/significance: eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs. These data delimit the end of the developmental window during which paternal rescue is still possible. Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis.

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Pollen grains prior to germination.(A–C) Mature pollen grains were stained with Alexander's stain to check for viability. (A) wild type, (B) eif3h-1, and (C) eif3e-1 mutant. (D–F) Mature pollen tetrads were stained with DAPI to visualize the nuclei. (D) wild type, (E) eif3h-1, and (F) eif3e-1 mutant. The location of the vegetative nucleus (dotted outline) was confirmed by refocusing when it was not in the same plane as the two sperm nuclei. Genotypes of the anther bearing plants are indicated.
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pone-0017640-g003: Pollen grains prior to germination.(A–C) Mature pollen grains were stained with Alexander's stain to check for viability. (A) wild type, (B) eif3h-1, and (C) eif3e-1 mutant. (D–F) Mature pollen tetrads were stained with DAPI to visualize the nuclei. (D) wild type, (E) eif3h-1, and (F) eif3e-1 mutant. The location of the vegetative nucleus (dotted outline) was confirmed by refocusing when it was not in the same plane as the two sperm nuclei. Genotypes of the anther bearing plants are indicated.

Mentions: Plants harboring the recessive quartet1 (qrt1-2) mutation generate pollen grains that remain joined to each other in meiotic tetrads after anthesis because of an underlying genetic defect in a pectin methylesterase [32]. Plants of the genotype eif3h−/eIF3h+; FTL567/+; qrt1-2/qrt1-2 were generated using the crossing scheme shown in Fig. 1. As controls, we also selected FTL-tagged plants that were wild-type for QRT1. FTL-tagged eIF3h heterozygotes produced pollen tetrads in which two grains were clearly fluorescent (i.e. eIF3h+) and two were non-fluorescent (eif3h−) (Fig. 2A). Deviations from the 2∶2 segregation were rarely observed, indicating that the LAT52:eYFP transgene in FTL567 is expressed reliably and does not become spontaneously silenced. To address the possibility of false negative fluorescence data (i.e. eIF3h+;FTL567 pollen that nevertheless appeared non-fluorescent), we also examined pollen from plants that were homozygous for eIF3h+;FTL567. In these plants only between 0% and 3% of pollen grains appeared YFP-negative, a low percentage. The four pollen grains from plants segregating eif3h− and eIF3h+ were morphologically indistinguishable, and all four were routinely scored as viable using Alexander's stain (Fig. 3A, B). DAPI staining of the wild-type pollen grains revealed one weakly stained vegetative nucleus and two brightly stained sperm nuclei, as expected. The three nuclei reside close to each other in the center of the pollen grain, forming the male germ unit [33]. The same results were obtained for the eif3h-1 mutant pollen grains (Fig. 3D, E). Thus, the eif3h mutation causes no dramatic defect during the first stage of post-meiotic pollen development, the stage that encompasses the two subsequent mitotic divisions that result first in one vegetative nucleus and then the two sperm nuclei [34].


Fluorescence-tagged transgenic lines reveal genetic defects in pollen growth--application to the eIF3 complex.

Roy B, Copenhaver GP, von Arnim AG - PLoS ONE (2011)

Pollen grains prior to germination.(A–C) Mature pollen grains were stained with Alexander's stain to check for viability. (A) wild type, (B) eif3h-1, and (C) eif3e-1 mutant. (D–F) Mature pollen tetrads were stained with DAPI to visualize the nuclei. (D) wild type, (E) eif3h-1, and (F) eif3e-1 mutant. The location of the vegetative nucleus (dotted outline) was confirmed by refocusing when it was not in the same plane as the two sperm nuclei. Genotypes of the anther bearing plants are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3049774&req=5

pone-0017640-g003: Pollen grains prior to germination.(A–C) Mature pollen grains were stained with Alexander's stain to check for viability. (A) wild type, (B) eif3h-1, and (C) eif3e-1 mutant. (D–F) Mature pollen tetrads were stained with DAPI to visualize the nuclei. (D) wild type, (E) eif3h-1, and (F) eif3e-1 mutant. The location of the vegetative nucleus (dotted outline) was confirmed by refocusing when it was not in the same plane as the two sperm nuclei. Genotypes of the anther bearing plants are indicated.
Mentions: Plants harboring the recessive quartet1 (qrt1-2) mutation generate pollen grains that remain joined to each other in meiotic tetrads after anthesis because of an underlying genetic defect in a pectin methylesterase [32]. Plants of the genotype eif3h−/eIF3h+; FTL567/+; qrt1-2/qrt1-2 were generated using the crossing scheme shown in Fig. 1. As controls, we also selected FTL-tagged plants that were wild-type for QRT1. FTL-tagged eIF3h heterozygotes produced pollen tetrads in which two grains were clearly fluorescent (i.e. eIF3h+) and two were non-fluorescent (eif3h−) (Fig. 2A). Deviations from the 2∶2 segregation were rarely observed, indicating that the LAT52:eYFP transgene in FTL567 is expressed reliably and does not become spontaneously silenced. To address the possibility of false negative fluorescence data (i.e. eIF3h+;FTL567 pollen that nevertheless appeared non-fluorescent), we also examined pollen from plants that were homozygous for eIF3h+;FTL567. In these plants only between 0% and 3% of pollen grains appeared YFP-negative, a low percentage. The four pollen grains from plants segregating eif3h− and eIF3h+ were morphologically indistinguishable, and all four were routinely scored as viable using Alexander's stain (Fig. 3A, B). DAPI staining of the wild-type pollen grains revealed one weakly stained vegetative nucleus and two brightly stained sperm nuclei, as expected. The three nuclei reside close to each other in the center of the pollen grain, forming the male germ unit [33]. The same results were obtained for the eif3h-1 mutant pollen grains (Fig. 3D, E). Thus, the eif3h mutation causes no dramatic defect during the first stage of post-meiotic pollen development, the stage that encompasses the two subsequent mitotic divisions that result first in one vegetative nucleus and then the two sperm nuclei [34].

Bottom Line: We also detected reduced pollen germination for eif3e.However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation. eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs.Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee, United States of America.

ABSTRACT

Background: Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3) cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. To accomplish this we have developed a broadly applicable method to track mutant alleles that are not already tagged by a visible marker gene through the male lineage of Arabidopsis.

Methodology/principal findings: Fluorescence tagged lines (FTLs) harbor a transgenic fluorescent protein gene (XFP) expressed by the pollen-specific LAT52 promoter at a defined chromosomal position. In the existing collection of FTLs there are enough XFP marker genes to track nearly every nuclear gene by virtue of its genetic linkage to a transgenic marker gene. Using FTLs in a quartet mutant, which yields mature pollen tetrads, we determined that the pollen transmission defect of the eif3h-1 allele is due to a combination of reduced pollen germination and reduced pollen tube elongation. We also detected reduced pollen germination for eif3e. However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation.

Conclusion/significance: eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs. These data delimit the end of the developmental window during which paternal rescue is still possible. Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis.

Show MeSH
Related in: MedlinePlus