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Disruption of a gene for rice sucrose transporter, OsSUT1, impairs pollen function but pollen maturation is unaffected.

Hirose T, Zhang Z, Miyao A, Hirochika H, Ohsugi R, Terao T - J. Exp. Bot. (2010)

Bottom Line: Among the five SUT genes, OsSUT1 and OsSUT3 were found to be preferentially expressed and had temporal expression patterns that were distinct from each other.Expression of OsSUT1 in pollen was confirmed by a promoter-GUS fusion assay.However, starch accumulation during pollen development was not affected by disruption of OsSUT1, suggesting that the sugar(s) required for starch biosynthesis is supplied by other sugar transporters.

View Article: PubMed Central - PubMed

Affiliation: Hokuriku Research Center, National Agricultural Research Center, National Agriculture and Food Research Organization, Joetsu, Niigata 943-0193, Japan. dragon@affrc.go.jp

ABSTRACT
Sucrose transporters (SUTs) are known to play critical roles in the uptake of sucrose from the apoplast in various steps of sugar translocation. Because developing pollen is symplastically isolated from anther tissues, it is hypothesized that SUTs are active in the uptake of apoplastic sucrose into pollen. To investigate this possibility, a comprehensive expression analysis was performed for members of the SUT gene family in the developing pollen of rice (Oryza sativa L.) using real-time RT-PCR combined with a laser microdissection technique. Among the five SUT genes, OsSUT1 and OsSUT3 were found to be preferentially expressed and had temporal expression patterns that were distinct from each other. Expression of OsSUT1 in pollen was confirmed by a promoter-GUS fusion assay. The physiological function of OsSUT1 in pollen was further investigated using retrotransposon insertion mutant lines. While the homozygote of disrupted OsSUT1 (SUT1-/-) could not be obtained, heterozygote plants (SUT1+/-) showed normal grain filling. Their progeny segregated into SUT1+/- and SUT1+/+ with the ratio of 1:1, suggesting that the pollen disrupted for OsSUT1 is dysfunctional. This hypothesis was reinforced in vivo by a backcross of SUT1+/- plants with wild-type plants and also by in vitro pollen germination on the artificial media. However, starch accumulation during pollen development was not affected by disruption of OsSUT1, suggesting that the sugar(s) required for starch biosynthesis is supplied by other sugar transporters.

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Identification of the genotypes of individual pollen grain. (A) A pollen grain germinated on the agar medium was picked up with a fine pipette tip (marked in the circle). A magnified image of the pollen is lower left. (B) A typical result of genotyping by PCR, showing that seven out of eight pollen grains have wild type OsSUT1 (SUT1+) gene. A PCR product with the DNA template from the leaves of SUT1+/– plant was used as a control.
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fig5: Identification of the genotypes of individual pollen grain. (A) A pollen grain germinated on the agar medium was picked up with a fine pipette tip (marked in the circle). A magnified image of the pollen is lower left. (B) A typical result of genotyping by PCR, showing that seven out of eight pollen grains have wild type OsSUT1 (SUT1+) gene. A PCR product with the DNA template from the leaves of SUT1+/– plant was used as a control.

Mentions: To investigate further how OsSUT1 disruption affects the development and/or function of pollen, the total number of the pollen in an anther, the rate of matured pollen, and the pollen germination rate were examined using a disruption line NF2752. The total number of pollen grains in an anther and the percentage of matured pollen, as assessed by iodine staining, were not different, irrespective of the genotype (Fig. 4A). In another observation using all three disruption lines, the rate of matured pollen did not differ irrespective of the genotypes in each disruption line (see Supplementary Table S1 at JXB online). This suggests that a male gamete with disrupted OsSUT1 is able to develop into a mature pollen grain. This also implies that disruption of OsSUT1 does not affect pollen development, but rather some other pollen function(s) during the process of fertilization, for example, pollen germination or pollen tube elongation. Pollen germination in the mutants was assessed by an in vitro pollen germination assay carried out on agar medium. The percentage of germinated pollen was found to be significantly lower in SUT1+/– plants than in wild-type SUT1+/+ plants (Fig. 4B), suggesting that disruption of OsSUT1 does in fact impair pollen germination. Germinated pollen grains of SUT1+/– plants were then individually genotyped by PCR revealing that 35 out of 40 (88%) of the germinated pollen grains had a wild-type (SUT1+) genotype (Fig. 5; Table 5).


Disruption of a gene for rice sucrose transporter, OsSUT1, impairs pollen function but pollen maturation is unaffected.

Hirose T, Zhang Z, Miyao A, Hirochika H, Ohsugi R, Terao T - J. Exp. Bot. (2010)

Identification of the genotypes of individual pollen grain. (A) A pollen grain germinated on the agar medium was picked up with a fine pipette tip (marked in the circle). A magnified image of the pollen is lower left. (B) A typical result of genotyping by PCR, showing that seven out of eight pollen grains have wild type OsSUT1 (SUT1+) gene. A PCR product with the DNA template from the leaves of SUT1+/– plant was used as a control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2921200&req=5

fig5: Identification of the genotypes of individual pollen grain. (A) A pollen grain germinated on the agar medium was picked up with a fine pipette tip (marked in the circle). A magnified image of the pollen is lower left. (B) A typical result of genotyping by PCR, showing that seven out of eight pollen grains have wild type OsSUT1 (SUT1+) gene. A PCR product with the DNA template from the leaves of SUT1+/– plant was used as a control.
Mentions: To investigate further how OsSUT1 disruption affects the development and/or function of pollen, the total number of the pollen in an anther, the rate of matured pollen, and the pollen germination rate were examined using a disruption line NF2752. The total number of pollen grains in an anther and the percentage of matured pollen, as assessed by iodine staining, were not different, irrespective of the genotype (Fig. 4A). In another observation using all three disruption lines, the rate of matured pollen did not differ irrespective of the genotypes in each disruption line (see Supplementary Table S1 at JXB online). This suggests that a male gamete with disrupted OsSUT1 is able to develop into a mature pollen grain. This also implies that disruption of OsSUT1 does not affect pollen development, but rather some other pollen function(s) during the process of fertilization, for example, pollen germination or pollen tube elongation. Pollen germination in the mutants was assessed by an in vitro pollen germination assay carried out on agar medium. The percentage of germinated pollen was found to be significantly lower in SUT1+/– plants than in wild-type SUT1+/+ plants (Fig. 4B), suggesting that disruption of OsSUT1 does in fact impair pollen germination. Germinated pollen grains of SUT1+/– plants were then individually genotyped by PCR revealing that 35 out of 40 (88%) of the germinated pollen grains had a wild-type (SUT1+) genotype (Fig. 5; Table 5).

Bottom Line: Among the five SUT genes, OsSUT1 and OsSUT3 were found to be preferentially expressed and had temporal expression patterns that were distinct from each other.Expression of OsSUT1 in pollen was confirmed by a promoter-GUS fusion assay.However, starch accumulation during pollen development was not affected by disruption of OsSUT1, suggesting that the sugar(s) required for starch biosynthesis is supplied by other sugar transporters.

View Article: PubMed Central - PubMed

Affiliation: Hokuriku Research Center, National Agricultural Research Center, National Agriculture and Food Research Organization, Joetsu, Niigata 943-0193, Japan. dragon@affrc.go.jp

ABSTRACT
Sucrose transporters (SUTs) are known to play critical roles in the uptake of sucrose from the apoplast in various steps of sugar translocation. Because developing pollen is symplastically isolated from anther tissues, it is hypothesized that SUTs are active in the uptake of apoplastic sucrose into pollen. To investigate this possibility, a comprehensive expression analysis was performed for members of the SUT gene family in the developing pollen of rice (Oryza sativa L.) using real-time RT-PCR combined with a laser microdissection technique. Among the five SUT genes, OsSUT1 and OsSUT3 were found to be preferentially expressed and had temporal expression patterns that were distinct from each other. Expression of OsSUT1 in pollen was confirmed by a promoter-GUS fusion assay. The physiological function of OsSUT1 in pollen was further investigated using retrotransposon insertion mutant lines. While the homozygote of disrupted OsSUT1 (SUT1-/-) could not be obtained, heterozygote plants (SUT1+/-) showed normal grain filling. Their progeny segregated into SUT1+/- and SUT1+/+ with the ratio of 1:1, suggesting that the pollen disrupted for OsSUT1 is dysfunctional. This hypothesis was reinforced in vivo by a backcross of SUT1+/- plants with wild-type plants and also by in vitro pollen germination on the artificial media. However, starch accumulation during pollen development was not affected by disruption of OsSUT1, suggesting that the sugar(s) required for starch biosynthesis is supplied by other sugar transporters.

Show MeSH