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Loss of the Arabidopsis thaliana P₄-ATPase ALA3 reduces adaptability to temperature stresses and impairs vegetative, pollen, and ovule development.

McDowell SC, López-Marqués RL, Poulsen LR, Palmgren MG, Harper JF - PLoS ONE (2013)

Bottom Line: We also demonstrate that ala3 mutants have reduced fecundity resulting from a combination of decreased ovule production and pollen tube growth defects.In-vitro pollen tube growth assays showed that ala3 pollen germinated ∼2 h slower than wild-type and had approximately 2-fold reductions in both maximal growth rate and overall length.Together, these results support a model in which ALA3 functions to modify endomembranes in multiple cell types, enabling structural changes, or signaling functions that are critical in plants for normal development and adaptation to varied growth environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada, United States of America.

ABSTRACT
Members of the P4 subfamily of P-type ATPases are thought to help create asymmetry in lipid bilayers by flipping specific lipids between the leaflets of a membrane. This asymmetry is believed to be central to the formation of vesicles in the secretory and endocytic pathways. In Arabidopsis thaliana, a P4-ATPase associated with the trans-Golgi network (ALA3) was previously reported to be important for vegetative growth and reproductive success. Here we show that multiple phenotypes for ala3 knockouts are sensitive to growth conditions. For example, ala3 rosette size was observed to be dependent upon both temperature and soil, and varied between 40% and 80% that of wild-type under different conditions. We also demonstrate that ala3 mutants have reduced fecundity resulting from a combination of decreased ovule production and pollen tube growth defects. In-vitro pollen tube growth assays showed that ala3 pollen germinated ∼2 h slower than wild-type and had approximately 2-fold reductions in both maximal growth rate and overall length. In genetic crosses under conditions of hot days and cold nights, pollen fitness was reduced by at least 90-fold; from ∼18% transmission efficiency (unstressed) to less than 0.2% (stressed). Together, these results support a model in which ALA3 functions to modify endomembranes in multiple cell types, enabling structural changes, or signaling functions that are critical in plants for normal development and adaptation to varied growth environments.

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Diagram of ALA3 showing T-DNA disruptions.Filled boxes represent exons and open boxes represent introns. T-DNA insertions are represented with triangles and identified by ala3 allele numbers, allele accessions and itb allele numbers where appropriate. Arrows identify oligos used for PCR genotyping and point in the 5′ to 3′ direction. The primers corresponding to the T-DNA left-borders are 1343 (SALK) and 638 (SAIL). The left border junction of ala3-2 is: CTTGTGAATTATTAACTCCTGCTTCGAcaacttaataacacattgcggacg. Capital letters represent ALA3 DNA and lowercase letters represent T-DNA. *Isolated in this study. aPublished by Poulsen et al. [22]. bPublished by Zhang and Oppenheimer [26].
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pone-0062577-g001: Diagram of ALA3 showing T-DNA disruptions.Filled boxes represent exons and open boxes represent introns. T-DNA insertions are represented with triangles and identified by ala3 allele numbers, allele accessions and itb allele numbers where appropriate. Arrows identify oligos used for PCR genotyping and point in the 5′ to 3′ direction. The primers corresponding to the T-DNA left-borders are 1343 (SALK) and 638 (SAIL). The left border junction of ala3-2 is: CTTGTGAATTATTAACTCCTGCTTCGAcaacttaataacacattgcggacg. Capital letters represent ALA3 DNA and lowercase letters represent T-DNA. *Isolated in this study. aPublished by Poulsen et al. [22]. bPublished by Zhang and Oppenheimer [26].

Mentions: Three independent ala3 alleles were used in this study (Figure 1). The ala3-1 (SAIL_422_C12) and ala3-4 (SALK_082157) alleles were previously used by Poulsen et al. [22] and shown to have stunted growth phenotypes for roots and rosettes. The ala3–4 allele corresponds to the itb2–6 allele used by Zhang and Oppenheimer [26]. Here we expanded our analyses to include a third independent allele, ala3-2 (SAIL_748_D03). All three lines were backcrossed multiple times to minimize the presence of second site mutations. Lines used for ala3-1 and ala3-2 were shown to be segregating a single basta-resistance marker, which is encoded within the T-DNA (Table 1, female outcrosses). Expression of ALA3 transgenes have been shown to rescue the ala3 root, rosette [22] and trichome [26] phenotypes, providing evidence that the phenotypes are due to a loss or low levels of ALA3 expression.


Loss of the Arabidopsis thaliana P₄-ATPase ALA3 reduces adaptability to temperature stresses and impairs vegetative, pollen, and ovule development.

McDowell SC, López-Marqués RL, Poulsen LR, Palmgren MG, Harper JF - PLoS ONE (2013)

Diagram of ALA3 showing T-DNA disruptions.Filled boxes represent exons and open boxes represent introns. T-DNA insertions are represented with triangles and identified by ala3 allele numbers, allele accessions and itb allele numbers where appropriate. Arrows identify oligos used for PCR genotyping and point in the 5′ to 3′ direction. The primers corresponding to the T-DNA left-borders are 1343 (SALK) and 638 (SAIL). The left border junction of ala3-2 is: CTTGTGAATTATTAACTCCTGCTTCGAcaacttaataacacattgcggacg. Capital letters represent ALA3 DNA and lowercase letters represent T-DNA. *Isolated in this study. aPublished by Poulsen et al. [22]. bPublished by Zhang and Oppenheimer [26].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646830&req=5

pone-0062577-g001: Diagram of ALA3 showing T-DNA disruptions.Filled boxes represent exons and open boxes represent introns. T-DNA insertions are represented with triangles and identified by ala3 allele numbers, allele accessions and itb allele numbers where appropriate. Arrows identify oligos used for PCR genotyping and point in the 5′ to 3′ direction. The primers corresponding to the T-DNA left-borders are 1343 (SALK) and 638 (SAIL). The left border junction of ala3-2 is: CTTGTGAATTATTAACTCCTGCTTCGAcaacttaataacacattgcggacg. Capital letters represent ALA3 DNA and lowercase letters represent T-DNA. *Isolated in this study. aPublished by Poulsen et al. [22]. bPublished by Zhang and Oppenheimer [26].
Mentions: Three independent ala3 alleles were used in this study (Figure 1). The ala3-1 (SAIL_422_C12) and ala3-4 (SALK_082157) alleles were previously used by Poulsen et al. [22] and shown to have stunted growth phenotypes for roots and rosettes. The ala3–4 allele corresponds to the itb2–6 allele used by Zhang and Oppenheimer [26]. Here we expanded our analyses to include a third independent allele, ala3-2 (SAIL_748_D03). All three lines were backcrossed multiple times to minimize the presence of second site mutations. Lines used for ala3-1 and ala3-2 were shown to be segregating a single basta-resistance marker, which is encoded within the T-DNA (Table 1, female outcrosses). Expression of ALA3 transgenes have been shown to rescue the ala3 root, rosette [22] and trichome [26] phenotypes, providing evidence that the phenotypes are due to a loss or low levels of ALA3 expression.

Bottom Line: We also demonstrate that ala3 mutants have reduced fecundity resulting from a combination of decreased ovule production and pollen tube growth defects.In-vitro pollen tube growth assays showed that ala3 pollen germinated ∼2 h slower than wild-type and had approximately 2-fold reductions in both maximal growth rate and overall length.Together, these results support a model in which ALA3 functions to modify endomembranes in multiple cell types, enabling structural changes, or signaling functions that are critical in plants for normal development and adaptation to varied growth environments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada, United States of America.

ABSTRACT
Members of the P4 subfamily of P-type ATPases are thought to help create asymmetry in lipid bilayers by flipping specific lipids between the leaflets of a membrane. This asymmetry is believed to be central to the formation of vesicles in the secretory and endocytic pathways. In Arabidopsis thaliana, a P4-ATPase associated with the trans-Golgi network (ALA3) was previously reported to be important for vegetative growth and reproductive success. Here we show that multiple phenotypes for ala3 knockouts are sensitive to growth conditions. For example, ala3 rosette size was observed to be dependent upon both temperature and soil, and varied between 40% and 80% that of wild-type under different conditions. We also demonstrate that ala3 mutants have reduced fecundity resulting from a combination of decreased ovule production and pollen tube growth defects. In-vitro pollen tube growth assays showed that ala3 pollen germinated ∼2 h slower than wild-type and had approximately 2-fold reductions in both maximal growth rate and overall length. In genetic crosses under conditions of hot days and cold nights, pollen fitness was reduced by at least 90-fold; from ∼18% transmission efficiency (unstressed) to less than 0.2% (stressed). Together, these results support a model in which ALA3 functions to modify endomembranes in multiple cell types, enabling structural changes, or signaling functions that are critical in plants for normal development and adaptation to varied growth environments.

Show MeSH
Related in: MedlinePlus