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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 tube lengths in eif3h and eif3e mutants.Pollen tube lengths were measured for one hundred non-fluorescent germinated pollen grains of (A) eif3h-1 and (B) eif3e-1 and compared with one hundred fluorescent (bona fide wild-type) pollen tubes. Data from two independent replicates were pooled. The difference in mean length was significant for eif3h (P<10−6, t-test), but not for eif3e (P>0.05).
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pone-0017640-g005: Pollen tube lengths in eif3h and eif3e mutants.Pollen tube lengths were measured for one hundred non-fluorescent germinated pollen grains of (A) eif3h-1 and (B) eif3e-1 and compared with one hundred fluorescent (bona fide wild-type) pollen tubes. Data from two independent replicates were pooled. The difference in mean length was significant for eif3h (P<10−6, t-test), but not for eif3e (P>0.05).

Mentions: When pollen from eif3h−/eIF3h+;FTL567/+ heterozygotes was germinated in pollen germination medium, the non-fluorescent (i.e. eif3h mutant) pollen germinated at a much reduced frequency compared to fluorescent (i.e. eIF3h+ wild-type pollen (Fig. 4A, B, Table 1). Moreover, the mutant pollen tubes were shorter than wild type (Fig. 5A). However, no significant aberration in pollen tube morphology (bifurcated pollen tubes, bulbous tip, etc.) was observed. Taken together, these analyses indicate that the developmental defect of the eif3h mutant male gametophyte occurs as a result of reduced germination frequency (∼1/4 of wild type) and reduced pollen tube growth (∼1/2 of wild type). We did not examine eif3h pollen growth in the natural environment of the pistil's transmitting tract. However, reciprocal crosses between eif3h and wild type showed a reduced transmission of the eif3h− mutant allele (2.6% [14]). Considering the poor transmission and the in vitro pollen growth defects described here, we surmise that eif3h pollen growth is compromised in the pistil as well.


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 tube lengths in eif3h and eif3e mutants.Pollen tube lengths were measured for one hundred non-fluorescent germinated pollen grains of (A) eif3h-1 and (B) eif3e-1 and compared with one hundred fluorescent (bona fide wild-type) pollen tubes. Data from two independent replicates were pooled. The difference in mean length was significant for eif3h (P<10−6, t-test), but not for eif3e (P>0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017640-g005: Pollen tube lengths in eif3h and eif3e mutants.Pollen tube lengths were measured for one hundred non-fluorescent germinated pollen grains of (A) eif3h-1 and (B) eif3e-1 and compared with one hundred fluorescent (bona fide wild-type) pollen tubes. Data from two independent replicates were pooled. The difference in mean length was significant for eif3h (P<10−6, t-test), but not for eif3e (P>0.05).
Mentions: When pollen from eif3h−/eIF3h+;FTL567/+ heterozygotes was germinated in pollen germination medium, the non-fluorescent (i.e. eif3h mutant) pollen germinated at a much reduced frequency compared to fluorescent (i.e. eIF3h+ wild-type pollen (Fig. 4A, B, Table 1). Moreover, the mutant pollen tubes were shorter than wild type (Fig. 5A). However, no significant aberration in pollen tube morphology (bifurcated pollen tubes, bulbous tip, etc.) was observed. Taken together, these analyses indicate that the developmental defect of the eif3h mutant male gametophyte occurs as a result of reduced germination frequency (∼1/4 of wild type) and reduced pollen tube growth (∼1/2 of wild type). We did not examine eif3h pollen growth in the natural environment of the pistil's transmitting tract. However, reciprocal crosses between eif3h and wild type showed a reduced transmission of the eif3h− mutant allele (2.6% [14]). Considering the poor transmission and the in vitro pollen growth defects described here, we surmise that eif3h pollen growth is compromised in the pistil as well.

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