Arabidopsis SHY2/IAA3 inhibits auxin-regulated gene expression - PubMed (original) (raw)

Comparative Study

Arabidopsis SHY2/IAA3 inhibits auxin-regulated gene expression

Qing Tian et al. Plant Cell. 2002 Feb.

Abstract

In Arabidopsis, SHY2 encodes IAA3, a member of the auxin-induced Aux/IAA family. Gain-of-function mutations in SHY2/IAA3 cause enlarged cotyledons, short hypocotyls, and altered auxin-regulated root development. Here we show that the gain-of-function mutation shy2-2 decreases both the induction and repression of auxin-regulated genes, suggesting that SHY2/IAA3 acts as a negative regulator in auxin signaling. shy2-2 affects auxin induction of many previously characterized primary response genes, implying that it might repress primary auxin responses. In addition, shy2-2 also affects expression of multiple auxin-nonresponsive genes. Light regulates expression of SHY2/IAA3, suggesting a possible link between light and auxin response pathways.

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Figures

Figure 1.

Figure 1.

RNA Gel Blot Analysis of Expression of Aux/IAA Genes in SHY2 (Wild Type) and shy2-2 and shy2-24 Mutants. Poly(A)+ RNA (5 μg) was isolated from 6-day-old SHY2, shy2-2, and shy2-24 light-grown seedlings that had been either mock treated (−) or treated with 20 μM IAA for 2 hr (+). RNA was separated in 1% agarose, transferred to a nylon membrane, and hybridized with 32P-labeled Aux/IAA probes or with a 32P-labeled β-tubulin probe. shy2-2 and shy2-24 were assayed in different experiments, and the wild-type controls for each experiment are shown. Exposure times were 72 hr for IAA1, IAA2, IAA4, IAA11, IAA12, IAA16, and IAA17 and 288 hr for IAA3, IAA5, IAA6, IAA7, IAA8, and IAA18.

Figure 2.

Figure 2.

Histochemical Analysis of Expression of _P_SHY2/IAA3::GUS. (A) X-gluc staining of 9-day-old wild type (WT), shy2-2, shy2-24, and _axr2-1 P_SHY2/IAA3::GUS seedlings grown in the light or in the dark. (B) 4-Methylumbelliferyl β-

d

-glucuronide (4-MU) assays of light-grown and dark-grown wild-type (WT), shy2-2, shy2-24, and _axr2-1 P_SHY2/IAA3::GUS seedlings.

Figure 2.

Figure 2.

Histochemical Analysis of Expression of _P_SHY2/IAA3::GUS. (A) X-gluc staining of 9-day-old wild type (WT), shy2-2, shy2-24, and _axr2-1 P_SHY2/IAA3::GUS seedlings grown in the light or in the dark. (B) 4-Methylumbelliferyl β-

d

-glucuronide (4-MU) assays of light-grown and dark-grown wild-type (WT), shy2-2, shy2-24, and _axr2-1 P_SHY2/IAA3::GUS seedlings.

Figure 3.

Figure 3.

Inhibition of SHY2/IAA3 Expression by Light in the Presence of Sucrose. Six-day-old dark-grown wild-type seedlings were either given a pulse of red light (R) and then returned to darkness (D) or shifted to continuous white light (W). mRNA was isolated after 4 hr, and RNA gel blots were hybridized with SHY2/IAA3 cDNA, CAB, or rDNA probes. Exposure times were 1 hr (CAB and rDNA) and 72 hr (SHY2/IAA3).

Figure 4.

Figure 4.

Induction of SHY2/IAA3 Expression by Light in the Absence of Sucrose. Six-day-old dark-grown seedlings were given a pulse of red light and returned to darkness for 0, 2, or 4 hr. RNA was isolated as described in Methods, and RNA gel blots were hybridized with SHY2/IAA3, CAB, and rDNA probes. Exposure times were 1 hr (CAB and rDNA) and 360 hr (SHY2/IAA3).

Figure 5.

Figure 5.

Analysis of Aux/IAA Gene Expression in SHY2 (Wild-Type), shy2-2, and shy2-24 Seedlings. Six-day-old light-grown seedlings were either mock-treated (open bars) or treated with 20 μM IAA (closed bars) for 2 hr. Total RNA was isolated, and the gene chip experiment was performed as described in Methods. The expression levels (y axis) were computed using Affymetrix software and represent differential hybridizations between perfectly matched and mismatched oligodeoxyribonucleotides.

Figure 6.

Figure 6.

Histochemical Analysis of _P_AXR2/IAA7::GUS Expression. X-gluc staining of 9-day-old light-grown ([A] to [F], [J], and [K]) and dark-grown ([G] to [I] and [L]) wild-type ([A], [D], [G], [J], and [L]), shy2-2 ([B], [E], [H], and [K]), and shy2-24 ([C], [F], and [I]) _P_AXR2/IAA7::GUS seedlings. (A) to (C) and (G) to (I) Shoot meristems. (D) to (F) Primary root tips. (J) Lateral root tips. (K) Cotyledons and leaves. (L) Hypocotyl/root junction. Bar in (A) = 0.25 mm for (A) to (I); bar in (J) = 0.15 mm; and bars in (K) and (L) = 0.5 mm.

Figure 7.

Figure 7.

Analysis of SAUR (A), GH3 (B), and ACS (C) Gene Expression in SHY2 (Wild-Type), shy2-2, and shy2-24 Seedlings. Six-day-old light-grown seedlings were either mock treated (−IAA) or treated with 20 μM IAA (+IAA) for 2 hr. Total RNA was isolated, and the gene chip experiment was performed as described in Methods. The expression levels (y axis) were computed using Affymetrix software and represent differential hybridizations between perfectly matched and mismatched oligodeoxyribonuleotides.

Figure 8.

Figure 8.

Histochemical Analysis of _P_SAUR-AC1::GUS Expression. (A) X-gluc staining of 9-day-old light- and dark-grown SHY2 (wild-type), shy2-2, and _shy2-23 P_SAUR-AC1::GUS seedlings. (B) 4-Methylumbelliferyl β-

d

-glucuronide (4-MU) assay of 9-day-old dark-grown SHY2 (wild-type), shy2-2, and _shy2-23 P_SAUR-AC1::GUS seedlings.

Figure 8.

Figure 8.

Histochemical Analysis of _P_SAUR-AC1::GUS Expression. (A) X-gluc staining of 9-day-old light- and dark-grown SHY2 (wild-type), shy2-2, and _shy2-23 P_SAUR-AC1::GUS seedlings. (B) 4-Methylumbelliferyl β-

d

-glucuronide (4-MU) assay of 9-day-old dark-grown SHY2 (wild-type), shy2-2, and _shy2-23 P_SAUR-AC1::GUS seedlings.

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