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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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A diagram showing the exon/intron structure of OsSUT1 gene and the position of the insertion of retrotransposon Tos17 in the three mutant lines used in this study. The arrowheads indicate the direction of Tos17 at the respective insertion sites.
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fig3: A diagram showing the exon/intron structure of OsSUT1 gene and the position of the insertion of retrotransposon Tos17 in the three mutant lines used in this study. The arrowheads indicate the direction of Tos17 at the respective insertion sites.

Mentions: To investigate the functions of OsSUT1 and OsSUT3 in pollen, gene disruption mutants of the two genes were searched for from a population of rice mutants generated by insertion of a retrotransposon Tos17. Three mutant lines of OsSUT1 were identified, but none were identified for OsSUT3. It was therefore decided to study OsSUT1 function in pollen further using three mutant lines, NC7083, NF8036, and NF2752, in which insertions of Tos17 occurred in exons 3, 4, and 10, respectively (Fig. 3). Populations of these three mutant lines segregated into two genotypes, homozygous wild-type (SUT1+/+) and heterozygous for the Tos17 insertion (SUT1+/–); however, no homozygous plants for the insertion (SUT1–/–) were identified (data not shown). Three hypotheses were developed to explain the absence of SUT1–/– plants: (i) both male and female gametes with disrupted OsSUT1 are functional, but the SUT1–/– zygote cannot develop to maturity, (ii) a male gamete is not formed, or is dysfunctional, and (iii) a female gamete is not formed or dysfunctional. To determine which of these three hypotheses is valid, the progeny of SUT1+/– plants were grown and investigated further. The germination frequency of the seeds from SUT1+/– plants ranged between 90% and 97% in four independent observations, which was not different from that of the seeds from SUT1+/+ plants for all three mutant lines. However, homozygous plants did not develop once again, and the segregation ratio of SUT1+/+ and SUT1+/– were 1:1 in all the three mutant lines (Table 2). The SUT1+/– plants grew normally and no differences were detected between SUT1+/+ in the major agronomic traits, including days to heading, plant height, or panicle number per plant (data not shown). In addition, both the total number of spikelets and the percentage of filled grains per panicle did not differ between the two genotypes (Table 3). If the first hypothesis were true, the segregation ratio of SUT1+/+:SUT1+/– should be 1:2, with one-quarter of the caryopses (SUT1–/– zygotes) being infertile or immature. If the third hypothesis were true, the segregation ratio should be 1:1, and half of the caryopses with SUT1- female gametes would be infertile. Thus, the second hypothesis appears to be valid; in this case, the segregation ratio should be 1:1, with the number of filled grains unaffected. To confirm this, SUT1+/– plants were backcrossed to wild-type plants. A total of 74 F1 seeds were obtained using SUT1+/– zygotes of the three insertion lines as pollen parents, but none of the F1 plants inherited the OsSUT1 mutant allele, indicating that pollen function is, in fact, impaired by disruption of the OsSUT1 gene (Table 4).


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)

A diagram showing the exon/intron structure of OsSUT1 gene and the position of the insertion of retrotransposon Tos17 in the three mutant lines used in this study. The arrowheads indicate the direction of Tos17 at the respective insertion sites.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: A diagram showing the exon/intron structure of OsSUT1 gene and the position of the insertion of retrotransposon Tos17 in the three mutant lines used in this study. The arrowheads indicate the direction of Tos17 at the respective insertion sites.
Mentions: To investigate the functions of OsSUT1 and OsSUT3 in pollen, gene disruption mutants of the two genes were searched for from a population of rice mutants generated by insertion of a retrotransposon Tos17. Three mutant lines of OsSUT1 were identified, but none were identified for OsSUT3. It was therefore decided to study OsSUT1 function in pollen further using three mutant lines, NC7083, NF8036, and NF2752, in which insertions of Tos17 occurred in exons 3, 4, and 10, respectively (Fig. 3). Populations of these three mutant lines segregated into two genotypes, homozygous wild-type (SUT1+/+) and heterozygous for the Tos17 insertion (SUT1+/–); however, no homozygous plants for the insertion (SUT1–/–) were identified (data not shown). Three hypotheses were developed to explain the absence of SUT1–/– plants: (i) both male and female gametes with disrupted OsSUT1 are functional, but the SUT1–/– zygote cannot develop to maturity, (ii) a male gamete is not formed, or is dysfunctional, and (iii) a female gamete is not formed or dysfunctional. To determine which of these three hypotheses is valid, the progeny of SUT1+/– plants were grown and investigated further. The germination frequency of the seeds from SUT1+/– plants ranged between 90% and 97% in four independent observations, which was not different from that of the seeds from SUT1+/+ plants for all three mutant lines. However, homozygous plants did not develop once again, and the segregation ratio of SUT1+/+ and SUT1+/– were 1:1 in all the three mutant lines (Table 2). The SUT1+/– plants grew normally and no differences were detected between SUT1+/+ in the major agronomic traits, including days to heading, plant height, or panicle number per plant (data not shown). In addition, both the total number of spikelets and the percentage of filled grains per panicle did not differ between the two genotypes (Table 3). If the first hypothesis were true, the segregation ratio of SUT1+/+:SUT1+/– should be 1:2, with one-quarter of the caryopses (SUT1–/– zygotes) being infertile or immature. If the third hypothesis were true, the segregation ratio should be 1:1, and half of the caryopses with SUT1- female gametes would be infertile. Thus, the second hypothesis appears to be valid; in this case, the segregation ratio should be 1:1, with the number of filled grains unaffected. To confirm this, SUT1+/– plants were backcrossed to wild-type plants. A total of 74 F1 seeds were obtained using SUT1+/– zygotes of the three insertion lines as pollen parents, but none of the F1 plants inherited the OsSUT1 mutant allele, indicating that pollen function is, in fact, impaired by disruption of the OsSUT1 gene (Table 4).

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