Os12g0601400

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OsIAA3 is one of IAA family members whose expression is rapidly increased in response to auxin.

Annotated Information

Function

  • The IAA gene, a well-studied gene in Arabidopsis that serves as a negative regulator of auxin signaling.
  • OsIAA3 is one of these family members whose expression is rapidly increased in response to auxin.


Expression

  • The researchers performed semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis using primers that specifically amplified the OsIAA3 3¢ non-coding region. Wild-type(WT) plants were treated with media containing 20 lM IAA. The OsIAA3 transcript was increased by IAA within 15–30 min,and reached a maximum within 1 h (Figure 2a).To confirm that OsIAA3 also interacts with Aux/IAA and ARF, we performed a yeast two-hybrid assay using OsIAA3 and OsARF1 (Sato et al., 2001; Waller et al., 2002). All transformed yeast cells formed colonies on the medium with histidine, while only yeasts expressing both OsIAA3 protein and OsIAA3 or OsIAA3 and OsARF1 protein formed colonies on the medium without histidine (Figure 2b). This indicates that OsIAA3 interacts with both OsIAA and OsARF proteins,as do Aux/IAA proteins in Arabidopsis.The researchers also analyzed the degradation of OsIAA3 protein produced in Escherichia coli with or without MG132 in the crude extract of rice seedlings. The level of OsIAA3 protein decreased within 1 h without MG132 treatment, while it remained after 8 h of treatment with MG132 (Figure 2c).
'Figure 2. Molecular analysis of the OsIAA3 gene.(a) Induction of OsIAA3 by auxin. Rice seedlings were treated with 20 lM IAA,and whole plants were sampled after the indicated time. The level of OsIAA3 transcript was analyzed using semiquantitative RT-PCR. OsACT1 was used as a control.(b) Interaction between OsIAA3–OsIAA3 and OsIAA3–OsARF1 in the yeast two-hybrid system. Yeast was grown on medium without leucine and tryptophan (control, –LW) or without leucine, tryptophan, and histidine()LWH).(c) Time-course of degradation of OsIAA3 protein produced in Escherichia coli in extracts from 9-day-old wild-type seedlings in the absence [1% (v/v) DMSO] or presence of 100 lM MG132 detected by an immunoblot analysis using the OsIAA3 antibody.'
  • The researchers used a steroid hormone-inducible system (Aoyama and Chua, 1997). The full length of mOsIAA3 was fused in frame with the glucocorticoid hormone-binding domain (GR) controlled under the rice ACT1 promoter (Figure 3a), and transformed into rice calli. To confirm whether the function of the transformed mOsIAA3 gene could be regulated by treatment with dexamethasone (DEX), we treated the transgenic plants with or without DEX for 18 h, and then with IAA for 2 h. We measured the transcript levels of OsIAA3 and the other auxin-inducible OsIAA gene members, OsIAA4 and OsIAA5.The transcript levels were measured using semiquantitative RT-PCR. All these genes were upregulated by the IAA treatment without DEX, whereas enhanced expression by IAA did not occur for treatment with DEX (Figure 3b).
'Figure 3. The structure of mOsIAA3-GR and the expression of endogenous OsIAA genes in transgenic plants carrying mOsIAA3-GR.(a) The construction of mOsIAA3-GR. Proline at position 58 of OsIAA3 was exchanged for leucine using PCR-based mutagenesis. The mutagenized OsIAA3 (mOsIAA3) was fused to a human glucocorticoid receptor domain(GR) in an in-frame manner and expressed under the control of the rice ACTIN1 promoter.(b) The induction of endogenous OsIAA3, OsIAA4 and OsIAA5 transcripts by IAA (20 lM) in the transgenic plants with or without dexamethasone (DEX) treatment. The RNA levels for each gene were analyzed using semiquantitative RT-PCR. The transcript level of OsACT1 is shown as a control.'


Internal structure

  • The mOsIAA3 transgenic plants also showed irregular arrangements of large and small vascular bundles in leaf primordia (Figure 7).The researchers found that xylem development, especially development of protoxylem, was defective, while the development of phloem was almost normal in mOsIAA3 transgenic rice(Figure 7). This suggests that the vascular patterning, especially xylem formation, was affected by the inhibition of the auxin signal in rice.
'Figure 7. Internal structure of wild-type (WT) and mOsIAA3 plants. (a,b) Cross-sections of leaf primordia at the shoot apex region of 1-month-old (a) WT and (b) mOsIAA3 seedlings. (b) In mOsIAA3, the midrib is not clearly visible.(a,d) Cross-sections of the middle of the fourth leaf blade in (c) WT and (d) mOsIAA3 plants. In (a–d), the bar is 100 lm, and in (c) an arrowhead indicates the midrib.(e,f) Cross-sections of the P5 stage of (e) WT and (f) mOsIAA3 plants. In (e) and (f), the bar is 50 lm, and in (f) an ectopic small vascular bundle is shown by an arrow. (g–j) Mature large vascular bundles in the (g,h) leaf blade and (i,j) sheath of (g,i) WT and (h,j) mOsIAA3 plants (g-j: bar, 25 lm). lv, large vascular bundle; sv, small vascular bundle; ae, lysigenous aerenchyma; px, protoxylem; mx, metaxylem; MSC, mestome sheath cell; BSC, bundle sheath cell; st, sieve tube; cc, companion cell;ph, phloem region'


Labs working on this gene

  • Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya, Aichi 464-8601, Japan


References

  • Abel, S., Nguyen, M.D. and Theologis, A.(1995) The PS-IAA4/5-like family of early auxin-inducible messenger-RNAs in Arabidopsis thaliana. J. Mol. Biol. 251, 533–549.


Structured Information