Limits...
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.

Show MeSH
(A) The iodine-stained cross-sections of the anthers at different developmental stages used in the real-time RT-PCR. Bars=0.1 mm. (B) Transcript levels of OsSUT1 and OsSUT3 in the pollen at different developmental stages. The transcript levels the SUT genes determined by real-time RT-PCR were standardized against those of a constitutive ubiquitin gene RUBIQ1 and expressed on a logarithmic scale. Values are the means of three independent replications (bars indicate SE). See Materials and methods for the details of the developmental stage. (This figure is available in colour at JXB online.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: (A) The iodine-stained cross-sections of the anthers at different developmental stages used in the real-time RT-PCR. Bars=0.1 mm. (B) Transcript levels of OsSUT1 and OsSUT3 in the pollen at different developmental stages. The transcript levels the SUT genes determined by real-time RT-PCR were standardized against those of a constitutive ubiquitin gene RUBIQ1 and expressed on a logarithmic scale. Values are the means of three independent replications (bars indicate SE). See Materials and methods for the details of the developmental stage. (This figure is available in colour at JXB online.)

Mentions: To examine which of the five members of the rice SUT gene family is expressed in the developing pollen grains, real-time RT-PCR analysis combined with laser microdissection (LM) was employed. A typical image of LM and electrophoresis of a resultant RNA sample are shown in Supplementary Fig. S1 at JXB online. LM was performed using anthers chemically fixed with pure acetone; these preparations yielded higher quality RNAs than from the more commonly used ethanol/acetic acid fixative, although preservation of specimen structural integrity was somewhat inferior (data not shown). Because anthesis of rice glumaceous flowers occurs in an orderly manner within an inflorescence, the days to the anthesis (DTA) can be estimated for each spikelet according to their position in the panicle. Thus, the developmental stages of the pollen were classified into five classes according to the estimated DTA of each glumaceous flower, as follows: stage I (5–7 DTA), stage II (4–5 DTA), stage III (2–3 DTA), stage IV (1 DTA), and stage V (0 DTA). At stage I, starch was not detectable in the pollen by I2/KI staining, and the cellular remnant of the tapetum was evident in the anther (Fig. 1A). At stage II, starch began to accumulate in the pollen (Fig. 1A). By real-time RT-PCR, only two SUT genes, OsSUT1 and OsSUT3, were detectable in the pollen; mRNA for the other three SUT genes was very low, if at all (Fig. 1B). The temporal expression patterns of the two SUT genes were different; the mRNA level of OsSUT1 was not detectable in stage I, increased suddenly in stages II and III, and then decreased through stages IV and V; whereas expression of OsSUT3 was already detectable at stage I; it then increased and maintained the highest level during stages II to V.


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) The iodine-stained cross-sections of the anthers at different developmental stages used in the real-time RT-PCR. Bars=0.1 mm. (B) Transcript levels of OsSUT1 and OsSUT3 in the pollen at different developmental stages. The transcript levels the SUT genes determined by real-time RT-PCR were standardized against those of a constitutive ubiquitin gene RUBIQ1 and expressed on a logarithmic scale. Values are the means of three independent replications (bars indicate SE). See Materials and methods for the details of the developmental stage. (This figure is available in colour at JXB online.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: (A) The iodine-stained cross-sections of the anthers at different developmental stages used in the real-time RT-PCR. Bars=0.1 mm. (B) Transcript levels of OsSUT1 and OsSUT3 in the pollen at different developmental stages. The transcript levels the SUT genes determined by real-time RT-PCR were standardized against those of a constitutive ubiquitin gene RUBIQ1 and expressed on a logarithmic scale. Values are the means of three independent replications (bars indicate SE). See Materials and methods for the details of the developmental stage. (This figure is available in colour at JXB online.)
Mentions: To examine which of the five members of the rice SUT gene family is expressed in the developing pollen grains, real-time RT-PCR analysis combined with laser microdissection (LM) was employed. A typical image of LM and electrophoresis of a resultant RNA sample are shown in Supplementary Fig. S1 at JXB online. LM was performed using anthers chemically fixed with pure acetone; these preparations yielded higher quality RNAs than from the more commonly used ethanol/acetic acid fixative, although preservation of specimen structural integrity was somewhat inferior (data not shown). Because anthesis of rice glumaceous flowers occurs in an orderly manner within an inflorescence, the days to the anthesis (DTA) can be estimated for each spikelet according to their position in the panicle. Thus, the developmental stages of the pollen were classified into five classes according to the estimated DTA of each glumaceous flower, as follows: stage I (5–7 DTA), stage II (4–5 DTA), stage III (2–3 DTA), stage IV (1 DTA), and stage V (0 DTA). At stage I, starch was not detectable in the pollen by I2/KI staining, and the cellular remnant of the tapetum was evident in the anther (Fig. 1A). At stage II, starch began to accumulate in the pollen (Fig. 1A). By real-time RT-PCR, only two SUT genes, OsSUT1 and OsSUT3, were detectable in the pollen; mRNA for the other three SUT genes was very low, if at all (Fig. 1B). The temporal expression patterns of the two SUT genes were different; the mRNA level of OsSUT1 was not detectable in stage I, increased suddenly in stages II and III, and then decreased through stages IV and V; whereas expression of OsSUT3 was already detectable at stage I; it then increased and maintained the highest level during stages II to V.

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