Introduction

The plant hormone gibberellin (GA) controls diverse biological processes such as stem elongation, seed germination and flowering. Many GA-related mutants have been isolated from the model plant species Arabidopsis thaliana and rice (Oryza sativa L.), and the majority of the underlying genes have been found to encode enzymes that are related to GA metabolism or components of GA signaling ^{1, 2, 3, 4, 5, 6, 7}. Many GA mutants show dramatically reduced plant height and hence increased yield potential and lodging resistance of modern cereal varieties, thus greatly contributing to the success of the 'green revolution' ^{8, 9, 10}. Recently, GID1 (GIBBERELLIN INSENSITIVE DWARF 1) was identified as a soluble GA receptor in rice and Arabidopsis ^{11, 12, 13}.

In addition to components in GA signaling, endogenous GA levels regulated by the GA metabolism also have an important role in the control of plant development. For example, a mutation in the GA20ox gene (sd-1) resulted in decreased GA levels in rice, which has been attributed to the rice 'green revolution' gene ^{9, 10}. Moreover, ectopic expression of OsGA2ox1 under the control of the promoter of the GA biosynthesis gene GA3ox2 resulted in a semi-dwarf phenotype, suggesting the potential for high yield production in rice ¹⁴. Many GA-related genes are feedback or feedforward regulated by bioactive GAs, where GA20ox and GA3ox function in GA biosynthesis with feedback regulation, and GA2ox functions in GA catabolism with feedforward regulation by bioactive GAs; GA metabolism is also closely related to GA signaling ^{4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15}.

Bioactive GA₁, GA₄, their immediate precursors GA₂₀ and GA₉, and C20 GAs are deactivated by GA2-oxidases ^{4, 15, 16, 17, 18}. However, 2-oxidation is not the only mechanism by which bioactive GAs are deactivated. Our previous study has demonstrated that the rice Eui gene (ELONGATED UPPERMOST INTERNODE), also known as Eui1 ¹⁹, encodes a novel P450 monooxygenase that catalyzes 16,17-epoxidation of non-13-hydroxylated GAs to generate bio-inactive 16,17-[OH]₂-GAs, representing a novel GA deactivation mechanism in rice ²⁰. The 16,17-[OH]₂-GAs were also found in extracts of other plant species, suggesting that 16,17-epoxidation might participate in GA deactivation by EUI-related enzymes in a variety of plants ²⁰. The finding that Eui is a GA catabolism gene provides additional evidence that the GA metabolism pathway is a useful target for increasing the agronomic value of crops. Indeed, the eui mutants have been used to genetically improve the heading performance of rice male sterile cultivars in hybrid rice production ^{21, 22, 23, 24, 25}.

The elevated expression of the Eui gene dramatically reduces the GA levels, leading to severe dwarf phenotypes in most transgenic plants that constitutively express Eui ²⁰. Therefore, fine-tuning EUI levels by either ectopic expression or RNA interference (RNAi) could effectively decrease or increase internode elongation, resulting in dwarfed or elongated transgenic plants for different breeding purposes. Interestingly, eui plants are still sensitive to exogenous GA₃ application, despite the highly accumulated levels of endogenous bioactive GAs in the mutants ^{20, 22}. Further experiments are needed to define the details of the mechanisms by which Eui regulates GA homeostasis and thereby GA signaling and other GA-related biological processes.

In this study, we investigated the regulation of Eui gene expression and its role in rice development. We generated transgenic plants that ectopically expressed Eui under the GA20ox2 (GA20ox-Eui) and GA3ox2 (GA3ox-Eui) promoters, and decreased Eui expression through RNAi. We show that the Eui gene was induced by bioactive GAs, and that it was preferentially expressed in the vascular bundles of the elongating internodes. The GA signaling genes GID2, GID1 and SLR1 were repressed in the eui mutant and were upregulated in the Eui-OX transgenic plants. The expression of RAmy1A and GAmyb was highly elevated in the eui mutants compared with the wild type (WT), leading to higher -amylase activity in the aleurone layer. Accordingly, starch granule development was defective in the root-tip cells of the eui mutants, whereas it was enhanced in Eui-OX plants, resulting in altered gravity responses. Ectopic expression of the Eui gene driven by the GA gene promoters dramatically decreased plant height in a dose-dependent manner. We also discuss how the Eui gene is involved in GA homeostasis and its potential utilization in crop improvement.

Results

Differential response of eui mutants and WT plants to GAs

eui mutants accumulate extremely high levels of GA₁ and GA₄ ²⁰. However, our previous studies have shown that eui mutants remain highly sensitive to exogenous GA₃ application at the heading stage ²². To study the responsiveness of eui mutants to exogenous GAs at the seedling stage, we incubated eui-1 and WT plants with bioactive GA₃, GA₁, GA₄ and the precursor GA₉. As shown in Figure 1A and 1B, GA₃ was most effective in increasing plant height in both WT and eui plants. The eui mutants were significantly more responsive than the WT plants to GA₄ and GA₉. Because GA₄ and GA₉ are the substrates of EUI ²⁰, they would not be deactivated by a functional EUI and therefore exhibited greater activity in eui plants. Although both eui and WT plants responded to GA₁, no significant difference was observed in their responses to this GA molecule since the control eui plants were found to be slightly taller than WT plants (Figure 1A and 1B). Similar results were also observed with spray treatment of GAs (data not shown). We propose that rice may have a high saturated GA response threshold. In support of this idea, we found a saturated response to about 9 mM GA₃ in WT plants at the seedling stage (data not shown).

Figure 1.

GA sensitivity of eui and wild-type plants and GA induction of Eui. (A) Sensitivity of eui mutant and wild-type (WT) seedlings to different GA molecules. Two-week-old seedlings were incubated with GAs (1 M) or without GAs (mock) for 1 week. (B) Plant elongation of eui and wild-type seedlings treated with GAs. Plant height was measured at day 7 after GA incubation. Error bars show standard errors (SE). (C) GA induction of Eui. Wild-type seedlings were incubated with GA₁, GA₃ and GA₄ (1 M) for 0-12h. The RT-PCR products were subjected to DNA gel blot analysis to detect the Eui expression levels. Ubi-1 was used as a control. Results are representative of two independent experiments with similar results.

Full figure and legend (127K)

Since EUI uses GA₄ but not GA₁ as a substrate, and GA₃ is not an endogenous GA, we next tested whether these GAs differentially induce the Eui gene in seedling leaves. We found that Eui expression was induced by GA₄ within 1-4 h (Figure 1C). Eui was also induced by GA₁ and GA₃ in a biphasic pattern that is different from that of GA₄ (Figure 1C). This kind of transient GA induction of Eui is probably in agreement with its expression pattern of strong tissue and developmental specificity ²⁰. Our current study indicates that the Eui gene is also involved in GA homeostasis at the seedling stage (also see below), even though Eui is not expressed at high levels in young seedlings and there is no strong phenotype of the eui mutants at this stage ²⁰.

Eui is highly expressed in vascular cells

Eui is primarily expressed in rapidly elongating or dividing tissues, including the divisional zone and the node of the uppermost internode ²⁰. Bioactive GAs are thought to be produced in these regions because GA20ox2 and GA3ox2 are also predominantly expressed here ²⁶. To test whether Eui is expressed in specific cells in this region, we analyzed -glucuronidase (GUS) staining of cross-sections of the divisional zone of an elongating uppermost internode of the Eui-GUS transgenic plant ²⁰. Strong GUS staining was observed in the vascular bundles of the elongating internodes (Figure 2A). Interestingly, the highest level of GUS was found in the parenchyma cells (Figure 2B), suggesting that GAs might be produced in or transported through the vascular system.

Figure 2.

Cell-specific activity of the Eui promoter. (A) GUS staining was detected in the cross-section of the divisional zone of the elongating uppermost internode of the Eui-GUS transgenic plant. Bar=50 m. (B) The highest activity was found in the parenchyma cells (PC) of the vascular bundles (VB). Bar=200 m. V, vessel.

Full figure and legend (83K)

Eui regulates root starch granule development and gravity responses

An early study showed that GA plus kinetin treatment could remove starch granules in amyloplasts and therefore change gravisensitivity of cress roots ²⁷. We were interested in determining whether those Eui-mediated GA phenotypes affect starch accumulation and graviresponsiveness in rice roots. As shown in Figure 3A and 3B, starch granules were almost completely absent in eui root-tip cells, while their generation was enhanced in the roots of Eui-OX plants compared with WT plants. As a consequence of the altered starch granule development, we observed that Eui-OX roots were more hypersensitive than the WT to gravity. After 2 h of rotation away from vertical, most Eui-OX root tips bended near vertical (Figure 3C). All WT and Eui-OX root tips bended vertically when roots were rotated over 12 h. These results indicate that Eui is also involved in GA homeostasis in rice roots and reveal a novel role for GA in gravity responses.

Figure 3.

Effects of Eui on root starch granule development and gravity responses. (A) Starch granules in roots of eui mutant, wild-type and Eui-OX and transgenic parent (TP309) plants. Starch granules are stained as black spots in the root tips. Results are representative of three independent experiments with similar results. (B) Longitudinal section of stained seedling root tips. Starch granules are displayed as brown spots. Bar=100 m. (C) Gravisensitivity of seedling roots. Results are representative of two independent experiments with similar results. Bending angles of the majority of roots are indicated for each line. Bar=5 mm.

Full figure and legend (111K)

GA signaling is regulated by Eui expression

EUI P450 deactivates bioactive GAs and knockout mutants accumulate high levels of bioactive GAs ²⁰. Therefore, we next investigated whether the GA signaling genes are affected in eui and Eui-OX plants. As shown in Figure 4A, the expression of the positive GA signaling genes, the receptor gene GID1 ¹¹ and the F-box gene GID2 ⁵ was repressed in the eui mutant and increased in Eui-OX plants. Intriguingly, the negative GA regulator SLR1, which encodes a DELLA protein ³, was slightly repressed in the young panicle of eui plants compared with WT plants. Likewise, overexpression (OX) of Eui slightly increased SLR1 transcription compared with the WT. In support of our observation, SLR1 protein accumulation or stability was greater in transgenic plants constitutively expressing Eui ¹⁹. By contrast, D1, a positive regulator of GA signaling that encodes the -subunit of the heterotrimeric G protein ^{1, 2}, was not affected (Figure 4A). In addition, we observed that the GA biosynthesis genes GA3ox1 and GA20ox2 were downregulated and GA2ox1 was upregulated in the eui seedlings (Figure 4B), similar to their expression in the internode during the heading stage ²⁰. This result supports the above proposal that Eui also regulates GA homeostasis in the seedling stage.

Figure 4.

Effect of Eui expression on rice GA signaling. (A) Differential expression of GID1, GID2, SLR1 and D1 in eui mutant, wild-type and Eui-OX plants. Ubi-1 was used as a control during RT-PCR. Results are representative of two independent experiments with similar results. TP309 is the wild type for the Eui-OX transgene. (B) Transcript levels of the rice GA3ox1, GA20ox2, GA2ox1 and SLR1 in seedlings, as estimated by RT-PCR. Ubi-1 was used as a control. Results are representative of two independent experiments with similar results. (C) Amylase activity in the aleurone cells of the embryoless half-seeds of the wild type and eui mutant. Production of -amylase was detected by staining starch with iodine in plates with or without 1 M GA₃. Results are representative of two independent experiments with similar results. (D) Transcript levels of the rice RAmy1A, GAmyb and SLR1 in the eui mutant and wild-type aleurone cells with or without treatment with 1 M GA₃, as detected by RT-PCR. Ubi-1 was used as a control. Results are representative of two independent experiments with similar results.

Full figure and legend (138K)

Aleurone cells recognize GA signals and trigger expression of -amylase ²⁸. We therefore assayed amylase activity to further characterize the altered GA signaling in eui plants. WT and eui mutant embryoless half-seed plants were placed on starch plates with or without 1 M GA₃ for 3 days, and the starch was then stained with iodine. Production of -amylase from WT half-seeds was observed only on the plate containing GA₃. By contrast, eui half-seeds produced strong amylase activity even in the absence of exogenous GA₃ (Figure 4C). Similar production of -amylase was observed in the rice slender (slr) mutant ³. Consistently, we detected higher expression levels of the -amylase gene RAmy1A and GAmyb, a positive regulator of RAmy1A expression ²⁹, compared with their expression in the WT (Figure 4D). These results demonstrate that amylase activity was high in the eui seeds, which indicates that GA signaling is enhanced in the eui aleurone cells. However, we did not observe decreased SLR1 expression either in the eui aleurone cells or in the eui seedlings (Figure 4B and 4D), which suggests that SLR1 might detect the GA signal differentially in different rice tissues. Together, these results indicate that the GA signaling pathway is affected in eui and Eui-OX plants owing to altered GA homeostasis.

RNAi of Eui effectively increases internode elongation

The eui phenotype that has increased panicle exertion (heading performance) has been used in breeding for male sterile varieties of hybrid rice ^{21, 22, 23, 24, 25}. From this we developed rice that has an eui phenotype using RNAi. Using a double-stranded RNA (dsRNA) transgenic approach ³⁰, we efficiently generated Eui knockout/knockdown plants, of which more than 80% of independent transgenic plants showed an elongated internode phenotype with decreased or undetectable expression of Eui that is similar to the eui mutant (in Figure 5A-5C, the results from four representative T1 transgenic plants are shown). These RNAi lines were stably inherited within generations (data not shown). Consequently, this study provides a feasible approach to rapidly develop elite eui rice lines, which requires a long breeding term when using conventional breeding practices.

Figure 5.

Efficient knockout/knockdown of the Eui gene by RNAi. (A) Morphological phenotypes of RNAi (RNA interference) and wild-type seedlings (inset) and adult plants. Four representative independent RNAi plants (T1) are shown. Bar=10 cm. (B) Lengths of individual internodes of wild-type and RNAi plants. (C) Expression levels of Eui detected by RT-PCR. Ubi-1 was used as a control.

Full figure and legend (156K)

Ectopic expression of Eui strongly reduces plant height

The Eui gene dramatically reduces plant height when overexpressed in transgenic rice, leading to severe dwarfing and infertile transgenic plants ²⁰. We also observed rare Eui-OX transgenic plants that displayed different reductions in height and produced seeds but were unstable within generations (Supplementary information, Figure S1). We confirmed that the height reduction of these lines correlates well with expression levels of Eui (Supplementary information, Figure S1).

In order to exploit the Eui application in rice molecular breeding, we further transformed an elite but tall variety (R2212) with Eui under the control of the GA biosynthesis genes GA20ox2 and GA3ox2, which function in sequential synthesis of GA9/GA20 and GA₁/GA₄, respectively ⁴. As shown in Figure 6A (where results from four representative transgenic lines are shown), the independent transgenic lines expressing GA20ox-Eui exhibited a range of dwarf phenotypes with normal (lines 1 and 2) or less seed setting (lines 3 and 4), and later flowering. Their phenotypes were also dose-dependent on Eui expression (Figure 6B). Similarly, the transgenic plants with the GA3ox-Eui chimeric gene also exhibited plant height reduction in a dose-dependent manner. (In Figure 7A and 7C, the results from four representative T1 transgenic plants are shown.) In contrast to GA20ox-Eui plants, GA3ox-Eui plants showed less reduction in plant height, and some lines, such as line 1, exhibited a semi-dwarf phenotype with normal seed setting (Figure 7A and 7B). The different outcomes of the GA20ox-Eui and GA3ox-Eui transgenes are probably attributed to the tissue-specific expression patterns of GA3ox2 and GA20ox2 ²⁶. Similar results were also observed in transgenic plants expressing a GA3ox2-GA2ox1 chimera ¹⁴. The stable inheritance of these transgenic lines indicates the feasibility of genetic improvement of rice varieties by modulating GA catabolism with the Eui gene. These results also confirm that Eui is a strong regulator of GA homeostasis, which fine-tunes rice plant height in WT plants.

Figure 6.

Ectopic expression of Eui under the GA20ox2 promoter in a tall variety, R2212. (A) Morphological phenotypes of wild-type R2212 and GA20ox2-Eui seedlings (inset) and adult plants. Four representative independent transgenic plants are shown. Bar=10cm. (B) Expression levels of Eui detected by RT-PCR in GA20ox2-Eui plants. Ubi-1 was used as a control.

Full figure and legend (85K)

Figure 7.

Ectopic expression of Eui under the GA3ox2 promoter in a tall variety, R2212. (A) Morphological phenotypes of wild-type R2212 and GA3ox2-Eui seedlings (insert) and adult plants. Four representative independent transgenic plants (T1) are shown. Bar=10 cm. (B) Lengths of panicles and internodes of wild-type and transgenic plants. (C) Expression levels of Eui detected by RT-PCR in GA3ox2-Eui plants. Ubi-1 was used as a control.

Full figure and legend (135K)

Discussion

Fine-tuning of GA homeostasis is essential for the establishment of GA-related phenotypes ^{4, 7, 15}. The amount of bioactive GAs is tightly maintained by both GA synthesis and catabolism. At least in rice, GA catabolism is performed by two types of enzymes: GA2-oxidases that convert bioactive GA₁, GA₄, their immediate precursors and C20 GAs into inactive GAs by 2-hydroxylation; and EUI P450 that converts bioactive GA₄ and its precursor GA₉ into inactive 16,17 epoxy-GAs by 16,17-epoxidation. The Eui expression pattern and the eui phenotypes suggest that EUI is a major GA catabolism enzyme in internodes of rice at the heading stage ²⁰. We have shown that the eui mutant is more sensitive than the WT to GA₄ and GA₉ at the seedling stage, and both are EUI substrates (Figure 1A and 1B). We have further indicated that the expression of the Eui gene responds to GA₁, GA₃ and GA₄, although GA₁ is not an EUI substrate, and GA₃ is not an endogenous GA. We propose that because GA₁ and GA₃ are structurally similar to GA₄, they would exhibit a certain capacity to trigger the Eui promoter with a different pattern (Figure 1C), or, alternatively, that GA₁ and GA₃ regulate GA signaling and hence Eui expression (see below). In addition, starch granules were almost devoid in the root tip of the eui mutant and were increased in those of Eui-OX plants, consequently affecting root gravity tropism (Figure 3). Although treatment with GA plus kinetin removed starch granules and thereby altered the gravisensitivity of cress roots ²⁷, a reduction in the amount of GAs in maize seedlings does not significantly alter root graviresponsiveness ³¹. Our current study indicates that GA has a role in starch granule generation and gravity tropism in rice roots and suggests that plants might be different in their requirements for GA in graviresponsiveness. It will be interesting to investigate whether mutants related to GA signaling such as gid1, gid2, slr1 and d1 also alter gravity tropism. Although there is no significant eui phenotype and detectable GA₁ and GA₄ at the seedling stage ²⁰, we have found that the GA metabolism genes are indeed either feedback or feedforward regulated at this stage (Figure 4B). Collectively, our current study demonstrates that Eui is involved in GA homeostasis, not only at the heading stage but also at the seedling stage and in roots.

The Eui gene is also inducible by GA₃, a non-endogenous GA, although with a different pattern to induction by GA₄. Similar GA₃ induction was also observed for the Arabidopsis GA2ox genes ¹⁶. However, the rice OsGA2ox1 gene does not respond to exogenous GA₃ ³², suggesting that rice GA catabolism genes might differentially respond to this GA molecule. Detailed analysis of the structures and cis-elements of their promoters would provide further information about their regulation in GA responses. Interestingly, strong Eui-GUS expression was observed in vascular bundles (Figure 2). A similar cell-specific pattern was also observed for OsGA2ox1 ³². It is well established that GAs are produced in certain tissues, particularly in young panicles in rice ³³. However, there is no information about how bioactive GAs are delivered to other tissues/sites. Our current study raises the possibility that GAs might be transported through the vascular system.

Co-expression of GA biosynthesis and signaling genes in rice tissues indicates that the biosynthesis of active GAs occurs at the same site as GA signaling ²⁶. The Eui expression pattern also suggests that the sites of GA deactivation by EUI and the synthesis of bioactive GAs may partially overlap at the heading stage ²⁰. We have shown in this study that Eui is involved in GA homeostasis, and therefore modulates the expression of the genes that are involved in GA signaling and -amylase production (Figures 3 and 4). Embryoless half-seeds are used to eliminate the effect of the embryo (the site of GA production), so they are usually GA-deficient. Interestingly, the embryoless half-seeds of the eui mutant continued to produce and secrete large amounts of -amylase (Figure 4C). Because no GA biosynthesis is observed in the rice aleurone layer ²⁶, it is possible that the elevated GA levels in maturing eui seeds induce amylase expression and accumulation before the seed fully desiccates. An alternative explanation is that a trace amount of bioactive GAs might be present in the aleurone layer during seed development in the eui mutant so that once the seeds are imbibed they produce -amylase without GA treatment. In support of the first possibility, the GA2ox-deficient mutant M326 of barley accumulates high levels of GA₁, resulting in high levels of premature -amylase expression ³⁴.

Surprisingly, we found that the expression of both positive GA signaling genes GID1 and GID2 and the negative signaling gene SLR1 was repressed in the eui mutant internodes and increased in plants overexpressing Eui (Figure 4A). We propose that EUI influences the GA signaling network due to altered GA levels according to a feedback mechanism that is similar to that operating in GA biosynthesis. It is possible that the eui mutant plants accumulate high levels of bioactive GAs, and that the overproduction of bioactive GAs could feedback regulate GA signaling by decreasing the production of GID1 and GID2, since GA treatment reduced transcript abundance for all three GID1 genes of Arabidopsis ¹³. Such high GA levels could also repress SLR1 expression as SLR1 protein accumulation was decreased or eliminated by GA treatment ⁵. However, the proposed GA-mediated SLR1 transcription and relationship between these regulation events need more study ¹³, as SLR1 expression was not altered in the eui seedlings and aleurone cells compared with the WT in our current study.

Breeding cereal crops for a desirable plant height or morphology has been a goal of the agriculture industry. In rice, male sterile cultivars are commonly defective in elongation of the uppermost internode owing to inadequate GA production in the empty anthers ³⁵. The eui mutation provides a tool for genetically improving the heading performance of male sterile cultivars, and rice varieties carrying the eui mutation have been officially released in China ^{21, 22, 23, 24, 25}. However, traditional breeding of eui varieties takes a long time because of its recessive nature. We efficiently developed eui rice using an RNAi transgene (Figure 5), and we are currently using this approach to develop elite eui male sterile lines. On the other hand, dwarf architecture, or the so-called 'green revolution', has been valuable in rice breeding, which confers lodging resistance and high yield potential. Genetic manipulation of the levels of bioactive GA is a practical strategy to generating rice with suitable plant height for high yield. Two approaches can be adopted to lower endogenous GA levels and therefore develop elite varieties; these include decreasing GA biosynthesis and increasing GA catabolism. Indeed, by using the mutant GA biosynthesis gene sd-1, a mutated GA20ox gene ^{9, 10}, or by using the GA catabolism gene OsGA2ox1 to manipulate GA levels ¹⁴, breeding for semi-dwarf rice is now feasible through molecular design.

In our previous study, we developed Eui-OX transgenic plants that showed a severe dwarf phenotype with defects in flower development ²⁰, and the semi-dwarf plants generated were genetically unstable (Supplementary information, Figure S1), limiting the use of the constitutive expression approach in rice molecular breeding. We therefore generated transgenic plants of a tall variety with GA20ox-Eui and GA3ox-Eui fusions (Figures 6 and 7). In contrast to the GA20ox-Eui transgenic approach, the GA3ox-Eui fusion produced more desirable semi-dwarf lines with normal seed setting. The same GA3ox2 (D18) promoter was also used to drive GA2ox1 expression and to generate semi-dwarf plants ¹⁴. This approach can also be used in other cereals such as maize and barley. Regardless, this study suggests that manipulation of the Eui gene could improve rice varieties through the modulation of GA catabolism.

Materials and Methods

GA response assay

Two-week-old WT (ZS97) and eui-1 mutant plants were incubated in 1/2 MS medium containing 1 M GA₁, GA₃, GA₄ and GA₉; seedling height (from the base to the leaf tip) was measured at day 7 after GA treatment. For GA induction of Eui, seedlings were treated with 1 M GA₁, GA₃ and GA₄, and samples were collected at different time points for RNA isolation.

Amylase activity

Matured seeds of the WT and the eui-1 mutant were dried and stored for 3 months. Embryoless half-seeds were sterilized in 3% NaClO for 15 min, washed with sterile water three times, and then incubated on agar plates containing 0.2% starch with or without 1 M GA₃ for 3 days at 28 °C in darkness. The plates were exposed to iodine vapor to determine -amylase activity as described previously ³⁶. The same treated half-seeds were collected at day 2 for RNA preparation to detect RAmy1A, GAmyb and SLR1 expression.

GUS staining

Histochemical assays for GUS activity in transgenic plants were performed as described previously ³⁷. Transverse sections (4 m) of the divisional zone of the elongating uppermost internode of the Eui-GUS transgenic plant ²⁰ were observed and photographed under a microscope (Olympus, Tokyo, Japan).

Detection of starch granules and root gravitropism

Seedlings were grown in 1/2 MS medium. Root starch granule staining was performed as described previously ³⁸. Longitudinal sections (2 m) of stained root tips were examined and photographed under a microscope (Olympus, Tokyo, Japan). For gravitropism analysis, 2-week-old seedlings grown in 1/2 MS agar were rotated by 90°. The root tip positions were recorded at 2 h after rotation.

Transgenic constructs and rice transformation

OX of Eui has been described previously ²⁰. The entire RNAi cassette of sense and antisense fragments of Eui was cloned into the vector pCAMBIA1300 s (provided by Dr Yinong Yang) ³⁰, and was introduced into the WT variety TP309 to generate more than 25 independent plants by Agrobacterium tumefaciens-mediated transformation. Progeny plants (T1 up to T3) were assayed. For the ectopic expression of Eui, the promoter regions that were 2.0 kb upstream of the coding regions of GA20ox2 (Sd1, accession number AF465255) and 1.38 kb upstream of the coding regions of GA3ox2 (D18, accession number P0013F10.29) were fused to the full-length Eui cDNA (accession number AY987040) to generate GA20ox-Eui and GA3ox-Eui fusions. These were inserted into the vector pCAMBIA1300 (accession number AF234269). The plasmids were transformed into a tall japonica variety R2212 by Agrobacterium tumefaciens-mediated transformation to generate more than 30 independent transgenic lines for each construct. T0 (severe dwarf, no seed setting) or T1 (seed setting) progeny plants were analyzed.

RNA preparation and transcript analysis

Total RNA was isolated from treated tissues or transgenic plant stems using TRIzol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). The Eui transcripts were detected by RT-PCR and Southern blot as described previously ²⁰. The transcripts of RAmy1, GAmyb, GID1, GID2, SLR1 and D1 were detected by reverse transcriptase PCR (RT-PCR) with the following primers: Ramy-F 5'-CGCGTCGCACCGAAGCAGAGTA-3'; Ramy-R 5'-AGCAGAGCATCCAGCCCACA-3'; GAMB-F 5'-CATGTAATACTACGGTTCTTAGCC-3'; GAMB-R 5'-GAATCTGCTTTAGCGTCTGG-3'; GID1-F 5'-GAGGTCAACCGCAACGAGTGC-3'; GID1-R 5'-GCTGCCGCCGTGGAAGAATA-3'; GID2-F 5'-CGGGGAGGACCTGGTGTTCG-3'; GID2-R 5'-CCCCTCCATTCTTATCACTGTCATTT-3'; SLR1-F 5'-GGTGCGGCCAAGGATCGTCA-3'; SLR1-R 5'-AGGAGCGTGCTCGCCTGTTT-3'; D1-F 5'-AAGGAGGATGTGCTTTATG-3'; and D1-R 5'-TGGTCTAGGGCCGTAGTT-3', using the following PCR conditions: 94 °C for 4 min, followed by 28 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, and an elongation step at 72 °C for 10 min. The transcripts of GA20ox, GA3ox and GA2ox were detected as described previously ²⁰.

(Supplementary Information is linked to the online version of the paper on the Cell Research website.)

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Acknowledgements

We are grateful to Dr Shinjiro Yamaguchi (RIKEN, Japan) for critical reading of the manuscript, and to Professor Yinong Yang (Penn. State University, USA) for the rice RNAi vector. This work was supported by grants from the National Natural Science Foundation of China (30670186 and 30421001), and the Ministry of Science and Technology of China (2006AA10A102) to ZH.