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First published online March 16, 2007; 10.1104/pp.107.096586 Plant Physiology 144:121-133 (2007) © 2007 American Society of Plant Biologists The Rice YABBY1 Gene Is Involved in the Feedback Regulation of Gibberellin Metabolism1,[C],[W]National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China (M.D., Y.Z., Q.M., Y.H., Q.Z.); Institut de Biotechnologie des Plantes, Université Paris sud 11, Orsay F–91405, France (D.-X.Z.); and Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom (P.H.)
Gibberellin (GA) biosynthesis is regulated by feedback control providing a mechanism for GA homeostasis in plants. However, regulatory elements involved in the feedback control are not known. In this report, we show that a rice (Oryza sativa) YABBY1 (YAB1) gene had a similar expression pattern as key rice GA biosynthetic genes GA3ox2 and GA20ox2. Overexpression of YAB1 in transgenic rice resulted in a semidwarf phenotype that could be fully rescued by applied GA. Quantification of the endogenous GA content revealed increases of GA20 and decreases of GA1 levels in the overexpression plants, in which the transcripts of the biosynthetic gene GA3ox2 were decreased. Cosuppression of YAB1 in transgenic plants induced expression of GA3ox2. The repression of GA3ox2 could be obtained upon treatment by dexamethasone of transgenic plants expressing a YAB1-glucocorticoid receptor fusion. Importantly, we show that YAB1 bound to a GA-responsive element within the GA3ox2 promoter. In addition, the expression of YAB1 was deregulated in GA biosynthesis and signaling mutants and could be either transiently induced by GA or repressed by a GA inhibitor. Finally, either overexpression or cosuppression of YAB1 impaired GA-mediated repression of GA3ox2. These data together suggest that YAB1 is involved in the feedback regulation of GA biosynthesis in rice.
The plant hormone GA regulates development throughout the life cycle of the plant. The biosynthesis of GA is regulated by both developmental and environmental stimuli (for review, see Hedden and Phillips, 2000  ; Olszewski et al., 2002). The genes encoding GA-biosynthetic enzymes have been identified in numerous species and are most highly expressed in rapidly growing tissues or organs (Olszewski et al., 2002). The final steps in the biosynthesis of the bioactive GAs, from GA53 or GA12 to GA1 or GA4, respectively, are catalyzed in parallel pathways by GA 20-oxidase (GA20ox) and GA3ox, whereas the bioactive GA1 and GA4 and their immediate precursors (GA20 and GA9, respectively) are converted to inactive forms by GA2ox (Hedden and Phillips, 2000; Olszewski et al., 2002). These enzymes, which are particularly important for the control of bioactive GA levels, are encoded by small gene families (Hedden and Phillips, 2000; Sakamoto et al., 2004). Overexpression or suppression of GA20ox, GA3ox, or GA2ox genes in transgenic plants often results in altered concentrations of active GA, emphasizing the importance of these genes in regulating GA content (for review, see Hedden and Phillips, 2000). These genes are also important in GA homeostasis. Transcript levels of some GA3ox and GA20ox genes are reduced, while GA2ox transcripts are increased by elevated GA signaling or GA treatment (Hedden and Phillips, 2000; Olszewski et al., 2002), indicating that GA biosynthesis is controlled by feedback regulation through the activity of the GA response pathway. When this pathway is derepressed as a result of loss-of-function mutations in the DELLA proteins that act as negative regulators, bioactive GAs as well as GA20ox and/or GA3ox mRNA are present at lower levels than in the wild-type plants (Croker et al., 1990; Martin et al., 1996; Dill and Sun, 2001; Silverstone et al., 2001). Conversely, gain-of-function mutations in the repressors or loss-of-function mutations in the positive components of the GA response pathway often result in higher levels of bioactive GA and up-regulation of GA20ox and GA3ox gene expression (Fujioka et al., 1988; Talon et al., 1990; Xu et al., 1995; Cowling et al., 1998; Ueguchi-Tanaka et al., 2000; Amador et al., 2001).
No signaling components downstream of the DELLA proteins have been clearly identified as mediators of GA homoeostasis, although several transcriptional regulators have been shown to modify GA metabolic gene expression. For example, the KNOX I class of homeodomain proteins repress GA20ox gene expression in the shoot apical meristem (SAM) in a number of species (for review, see Hay et al., 2004
In rice (Oryza sativa), GA3ox and GA20ox are encoded by two and four genes, respectively (Sakamoto et al., 2004
The plant genome encodes a large number of transcription factors, many of which are specific to plants. Among them, the YABBY family, which is characterized by the juxtaposition of two conserved domains (a C2C2 zinc finger domain toward the amino terminus and a putative helix-loop-helix "YABBY" domain conserved in HMG and other transcription factors toward the carboxyl terminus), is not found in yeast or animal cells (Bowman and Smyth, 1999
The rice genome also seems to contain six members of the YABBY family. Recently, it has been shown that a rice CRC-related YABBY gene, DL (the mutation of which induces a drooping leaf phenotype), is required for carpel specification and leaf midrib development (Yamaguchi et al., 2004 In this work, we studied the expression pattern and regulatory function of rice YAB1 by transgenic approaches. Our data suggest that this gene is involved in the regulation of GA homeostasis in rice.
YAB1 Expression Pattern Reverse transcription (RT)-PCR analysis showed that YAB1 was expressed in both shoots and developing panicles (Fig. 1A ). In addition, YAB1 expression was detected in leaf sheaths at a lower level (Fig. 1A). In situ hybridization experiments detected YAB1 mRNA in the shoot apex and in young leaves (Fig. 1B, a and b). In the shoot apex, YAB1 mRNA was detected only in leaf primordia but not in the SAM (Fig. 1B, a). The expression of YAB1 in young leaves did not show any adaxial/abaxial polar distribution but was enhanced in both the upper and lower epidermis and in cells surrounding the vascular bundles (Fig. 1B, b). YAB1 mRNA was also detected by in situ hybridization in rachis meristems and in lemma, palea, stamen, carpel primordia, and the embryonic axis (Fig. 1B, c–e). No adaxial/abaxial polar distribution of YAB1 transcripts in the stamen or carpel primordia was observed (Fig. 1B, d). In addition, transgenic rice plants containing the GFP gene, under the control of the YAB1 promoter region spanning from the initiation ATG codon to –1,662, showed GFP expression in elongating internodes and in leaf sheaths (Fig. 1C, a and b), in which GFP was detected mostly in sclerenchyma, vascular bundles and epidermal cells (Fig. 1C, c and d), confirming the in situ hybridization data (Fig. 1B, b). Therefore, YAB1 was expressed widely in many different tissue or cell types, mostly in rapidly dividing or elongation tissues or organs but not in the SAM.
Overexpression of YAB1 Resulted in Semidwarf Phenotype To study the function of YAB1, full-length cDNAs of the gene were isolated from a cDNA library of indica rice ‘Minghui 63.’ The indica cDNA presented a number of nucleotide substitutions resulting in seven amino acid changes of the protein compared to the published japonica sequence (Supplemental Fig. S1). This cDNA was cloned between the double cauliflower mosaic virus (CaMV) 35S promoter and the NOS (nopaline synthase) terminator in a rice transformation vector (see "Materials and Methods"). The construct was introduced into ‘Nipponbare’ by Agrobacterium-mediated transformation, and more than 30 independent transgenic T0 plants were produced. A semidwarf phenotype occurred in many of the transgenic lines, with plant height about one-third that of the wild type together with about 3 times more tillers (Fig. 2A ; Tables I and II ). Further investigation of the semidwarf transgenic plants indicated that elongation of all the internodes, except internode V, was affected (Table I). Stem epidermal, elongating, and intercalary meristem cells in the transgenic plants were shorter than in the wild type (Fig. 2B), indicating that cell elongation was reduced. No alteration of leaf shape and flowering time was observed. The overexpression plants were partially fertile. Transgenic plants showing phenotypic changes were subsequently analyzed in the T1 generation for cosegregation between the phenotype and the transgene. Eight semidwarf transgenic plants were analyzed by RNA-gel blots. As shown in Figure 2C, all of the semidwarf plants overexpressed the transgene. Southern-blot analysis of these lines indicated that they contained a single transgene insertion in the genome (Supplemental Fig. S2).
The Dwarf Phenotype of YAB1-Overexpressing Plants Could Be Suppressed by Exogenous GA Treatment To determine whether or not the semidwarf phenotype of the YAB1 overexpression plants was caused by GA deficiency, the response of the plants to exogenous GA was measured. Four-week-old (at four-leaf stage) transgenic and wild-type plants were transferred to a hydroponic culture medium supplemented with 10 µM paclobutrazol (an inhibitor of GA biosynthesis) for 1 d to reduce the endogenous GA to background levels, before adding GA3 at different final concentrations ranging from 0.01 to 20 µM. Plant heights were measured 10 d after GA application. The data presented in Figure 3 showed that at 10 µM, GA could fully rescue the dwarf phenotype of the YAB1 overexpression plants.
Endogenous GA Contents in the Overexpression Plants
It has been shown that GA1 is the major bioactive GA in the vegetative organs in rice (Kobayashi et al., 1988
Overexpression of YAB1 Altered GA Metabolic Gene Expression
Changes in the GA content in the YAB1 overexpression lines may be due to altered expression of one or more of the genes encoding GA metabolic enzymes. We compared expression of six representative genes encoding four enzymes, ent-copalyl diphosphate synthase (CPS), GA3ox, GA20ox, and GA2ox, in young leaves of transgenic and wild-type plants by quantitative RT-PCR. As shown in Figure 4C, mRNA levels of GA3ox2 in the YAB1-overexpressing lines (Y1, Y3, and Y4) were reduced to 10% to 20% of the wild-type levels. In contrast, GA2ox3 was induced in the overexpression lines. It is suggested that GA3ox2 makes major contributions to the biosynthesis of GA1 during vegetative development (Kaneko et al., 2003
Examination of transgene copy numbers by Southern blots revealed that two lines had at least two insertions of the YAB1 transgene (Supplemental Fig. S2). These lines were found to contain lower YAB1 mRNA levels compared to the wild type (Supplemental Fig. S3A), suggesting that expression of both endogenous and transgenic YAB1 was being suppressed, as might be anticipated, because multiple copy transgenes have a high probability of inducing cosuppression and gene silencing (Schubert et al., 2004
The cosuppression plants did not show the slender phenotype, probably because of only slight increase of GA1 or effects on other developmental aspects (see below) induced by the cosuppression. Instead, leaf blades of the cosuppression plants drooped outwards, forming an acute angle with the leaf sheaths (Fig. 6, A and B ). In addition, leaves were rolled toward the abaxial side, forming a cylinder-like structure (Fig. 6D). Transverse sections showed increases in the number of the bulliform cells situated between the vasculatures on the adaxial side of the leaf blade (Fig. 6F). Bulliform cells deposited in the adaxial side of the leaf blade are responsible for the leaf rolling that may occur during drought stress in rice. These cells in Y8 plants had a rectangular or triangular shape rather than the spherical form observed in the wild-type plants. Other cells in the leaves appeared normal, with no alteration in adaxial-abaxial identity observed in the arrangement of the leaf inner structure. These observations suggested that the rolled leaf phenotype may be due to the increases in bulliform cell number and that YAB1 may have a function in bulliform cell division and differentiation. The cosuppression plants were sterile due to defects in anther and carpel formation (Fig. 6H).
YAB1 Is Directly Involved in the Regulation of GA3ox2
To determine whether YAB1 directly regulated the expression of the GA metabolic genes, we produced transgenic rice producing YAB1 fused with the glucocorticoid receptor (GR) that is used to sequestrate the fusion protein in the cytoplasm (Aoyama and Chua, 1997
To know whether YAB1 could bind to the GA3ox2 promoter, yeast one-hybrid assays revealed that that YAB1 interacted with a 0.8-kb promoter region of GA3ox2 (not shown). This promoter fragment was subsequently cut into two segments (Fig. 8A ) that were used for gel shift assays with Escherichia coli-produced YAB1 protein. The region from –662 to –329 was bound by YAB1 protein (Fig. 8B). Double-stranded oligonucleotides corresponding to seven overlapped regions of the DNA fragment were tested for binding to YAB1. One of the oligonucleotides (P3) retained the specific YAB1-binding activity (Fig. 8, C and D). Sequence analysis (with software PLANTCARE) revealed that this 50-bp region contained a GA-responsive element (GARE; 5'-TAACAGA; Fig. 8E). To test whether this element was responsible for the binding of YAB1, binding assays with oligonucleotides containing a pair of the wild-type or substituted GARE sequences showed that YAB1 bound specifically to the wild-type GARE (Fig. 8F).
These data together suggested a direct involvement of YAB1 in regulating the expression of GA3ox2.
The expression pattern of YAB1 in shoot and floral meristems or in elongating internodes was similar to that of GA biosynthetic genes (Fig. 1; Kaneko et al., 2003
We also investigated the effect of a low endogenous GA content on expression of YAB1 by treating wild-type plants with the GA biosynthesis inhibitor paclobutrazol. Plants were harvested for RNA extraction at the same time points as for the GA treatment, and YAB1 transcript was analyzed by real-time quantitative RT-PCR. As shown in Figure 9A, paclobutrazol treatment produced the opposite YAB1 expression profile as obtained after GA3 treatment.
To confirm the observations in Figure 9A, we tested the YAB1 mRNA levels in rice GA biosynthesis (eui) and signaling (gid1 and slr1) mutants. The eui mutants accumulate biologically active gibberellins and show repression of GA3ox2 (Zhu et al., 2006
Our results suggested that YAB1 may function as a negative regulator of GA3ox2. To know whether YAB1 played a role in mediating GA-dependent down-regulation of GA3ox2 expression, both YAB1 overexpression and cosuppression plants along with wild type at the four-leaf stage were sprayed with 50 µM GA3 and harvested at different time points for RNA extraction and quantitative RT-PCR analyses. In wild-type plants, the applied GA rapidly (1–2 h) suppressed the expression of GA3ox2 to a much lower level (about a 5-fold decrease). In YAB1 cosuppression plants (Y8), the applied GA had little effect on the expression of GA3ox2, suggesting that YAB1 is required for GA-mediated GA3ox2 repression. However, overexpression of YAB1 also attenuated the GA-induced repression of GA3ox2 (Fig. 10 ). The later observation could be explained by an already repressed expression level of GA3ox2 in YAB1 overexpression plants. Applied GA had less effect on its repression than in wild-type plants. These data suggested that YAB1 is required for the GA-mediated GA3ox2 repression.
In this work, we present several lines of evidence that YAB1 is involved in the feedback regulation of GA biosynthesis. In contrast to the SAM-expressed KNOX genes that exclude the expression of GA20ox from the SAM (for review, see Hay et al., 2004 ), the expression domains of YAB1 overlap with those of GA3ox2, GA20ox2, and GA-signaling genes, which are expressed in diverse organs or tissues of rice, including embryo, shoots, and floral meristems (Fig. 1; Kaneko et al., 2003). This suggests that YAB1 have a function in modulating GA metabolic gene expression or GA signaling throughout the plant. The semidwarf phenotype, which can be suppressed by applied GA, and the changes in GA metabolic gene expression and GA levels induced by both overexpression and cosuppression of YAB1 support a regulatory function for YAB1 in GA biosynthesis (Figs. 2–4 ). Importantly, the expression of YAB1 is GA responsive, being rapidly and transiently induced by GA or repressed by inhibition of GA biosynthesis (Fig. 9), suggesting that YAB1and GA form part of a regulatory loop in GA-responsive tissues. Finally, YAB1 binds to the promoter of GA3ox2 and is required for GA-mediated repression of the gene (Figs. 7, 8, and 10). These data together would support a feedback regulatory scenario; the rapid activation of YAB1 by GA subsequently induces the repression of GA3ox2 and consequently decreases bioactive GA levels, leading to the repression of YAB1 itself. In addition, the effects of overexpression and cosuppression of YAB1 on GA contents is relatively moderated (about 2-fold changes; Figs. 4 and 5) in favor of a feedback regulatory function of the gene in GA biosynthesis.
We have shown that overexpression of YAB1 induces repression of GA3ox2 and, consequently, accumulation of its substrate GA20 and a reduction in the amount of its bioactive product GA1 (Fig. 4). Accordingly, cosuppression of YAB1 results in increased GA3ox2 expression (Fig. 5). The experiments with the inducible YAB1 construct (Fig. 7) showed that the repression of GA3ox2 was obtained upon induction of the YAB1 activity. In addition, YAB1 interacts directly with a GARE sequence within the GA3ox2 promoter (Fig. 8). These data, together with the requirement of a normal expression level of YAB1 for GA-mediated GA3ox2 repression (Fig. 10), indicate that YAB1 is directly involved in the regulation of GA3ox2. In contrast, YAB1 overexpression induces GA2ox3, which is consistent with the accumulation of the 2-oxidized products GA8 and GA29 (Fig. 4). However, the cosuppression of YAB1 did not decrease the expression of GA2ox3 (Fig. 5; Supplemental Fig. S2), which helps to explain why there was only a slight increase of GA1 accumulation and there was no typical slender phenotype. This discrepancy suggests that YAB1 is not involved in GA2ox3 regulation at natural expression levels in wild-type plants or that there is a baseline level of GA2ox3 expression regulated independently of YAB1. It is also possible that a reduction of GA2ox3 expression in the absence of YAB1 might be compensated for by feedback induction due to the increase of the active GA1 in the cosuppression plants. However, failure to detect any YAB1-binding activity of the GA2ox3 promoter where no GARE was found (not shown) suggests that YAB1 is not directly involved in the regulation of the gene. Conversely, overexpression of YAB1 does not significantly alter GA20ox2 expression (Fig. 4), but cosuppression of YAB1 induces the expression of GA20ox2 (Fig. 5). We favor the hypothesis that the expression of GA20ox2 is indirectly induced in the cosuppression plants through an independent feedback mechanism required for coactivation of both of the key biosynthetic genes, GA3ox2 and GA20ox2, because there was no direct binding of YAB1 to GA20ox2 promoter that contains no GARE sequence (not shown). Therefore, our data show that YAB1 is a direct regulator of GA3ox2, but its deregulation has indirect impact on the other key GA metabolic gene expression.
GA biosynthesis is controlled by feedback regulation through the activity of the GA response pathway. DELLA proteins that act as negative regulators of the pathways are required for GA feedback regulation, as mutations in DELLA genes affect GA metabolic gene expression and bioactive GA homeostasis. GA induces DELLA protein degradation. It is suggested that DELLA proteins may directly or indirectly repress transcriptional activators of GA signaling pathways. However, no transcriptional regulator downstream of the DELLA proteins has been so far clearly identified as a mediator of GA homoeostasis, although it is shown that the tobacco (Nicotiana tabacum) protein REPRESSION OF SHOOT GROWTH (RSG), a bZIP transcription factor, binds to and activates the transcription of the early GA biosynthetic gene ent-kaurene oxidase. Overexpression of a dominant negative form of RSG results in a GA-deficient dwarf phenotype and prevents activation of an NtGA20ox gene in transgenic tobacco, suggesting that RSG plays a role in feedback regulation of NtGA20ox (Fukazawa et al., 2000
The GARE (TAACAGA) motif has been identified as a cis-regulatory element involved in the GA induction of rice
At this stage, it is not known whether the other members of YABBY proteins bind to GARE. Arabidopsis YABBY genes are expressed in the abaxial domain of the leaf and are required for specifying adaxial/abaxial polarity of the leaf, in addition to contributing to repression of KNOX1 expression in the leaf primordium. Previous results and data from this study have shown that the rice YABBY genes are not expressed in a polar manner and that deregulation of expression by mutation or by transgenics did not affect the leaf adaxial/abaxial polarity (Jang et al., 2004
It has to be pointed out that the YAB1 cDNA previous studied by Jang et al. (2004)
Materials
Rice (Oryza sativa spp. japonica) ‘Nipponbare’ was used in this study. The full-length cDNAs of YAB1 (accession no. AF098752) were isolated from a normalized cDNA library of the elite indica rice ‘Minghui 63’ (Zhang et al., 2005
The binary vector for transformation was constructed based on pCAMBIA1301 (CAMBIA) and pRTL2 (Mason et al., 1992
For expression analysis of YAB1, total RNA was extracted from different vegetative organs and panicles at different developmental stages using an RNA extraction kit (TRIzol Reagent, Invitrogen). For assaying expression of transgenes, total RNA was extracted from the flag leaves of the wild-type and transgenic plants. To quantify transcripts of GAox genes, total RNA was extracted from 1-month-old rice seedlings (four-leaf stage). For quantitative real-time PCR analysis, 4 µg total RNA was treated first with 2 U DNase I (Invitrogen) and then reverse transcribed in a total volume of 20 µL with 0.5 µg oligo(dT)15, 0.75 mM dNTPs, 10 mM dithiothreitol (DTT), and 200 U SuperScript II RNase H– reverse transcriptase (Invitrogen). RT-PCR was performed using gene-specific primers (see below) in a total volume of 25 µL with 1.5 µL of the RT reactions, 0.25 µM gene-specific primers, and 12.5 µL SYBR Green Master mix (Applied Biosystems) on a 7500 real-time PCR machine (Applied Biosystems) according to the manufacturer's instructions. The rice actin1 gene was used as the internal control. All primers were annealed at 58°C and run 45 cycles. The relative expression level of each gene in transgenic plants was compared to that in wild-type ones, after normalization using the actin1 cDNA level and averaging over three replicates. The primers used in RT-PCR for YAB1 were: YAB1-F (5'-CTTGCTCCTTTTCACCAAGC-3') and YAB1-R (5'-ATGAGCCCAGTTCTTTGCAG-3'). The primers used in GA synthetic gene expression analysis were: CPS1-F (5'-GCGTGCATTTTCGAACCAA-3'), CPS1-R (5'-TTGGCCAGCACTGACACTCT-3'); GA20ox1-F (5'-GCCACTACAGGGCCGACAT-3'), GA20ox1-R (5'-TGGTTGCAGGTGACGATGAT-3'); GA20ox2-F (5-'CCAATTTTGGACCCTACCGC-3'), GA20ox2-R (5'-GAGAGAAGCCCAACCCAACC-3'); GA3ox2-F (5'-TCCTCCTTCTTCTCCAAGCTCAT-3'), GA3ox2-R (5'-GAAACTCCTCCATCACGTCACA-3'); GA2ox1-F (5'-TGACGATGATGACAGCGACAA-3'), GA2ox1-R (5'-CCATAGGCATCGTCTGCAATT-3'); and GA2ox3-F (5'-TGGTGGCCAACAGCCTAAAG-3'), GA2ox3-R (5'-TGGTGCAATCCTCTGTGCTAAC-3'). The actin1 primers were: Actin1-F (5'-TGCTATGTACGTCGCCATCCAG-3') and Actin1-R (5'-AATGAGTAACCACGCTCCGTCA-3'). For semiquantitative RT-PCR analysis, 2 µg of total RNA was reverse-transcribed in a total volume of 20 µL with 0.5 µg oligo(dT)15, 0.75 mM dNTPs, 10 mM DTT, and 100 U SuperScript II RNase H– reverse transcriptase (Invitrogen). PCR was performed in a total volume of 20 µL with 1 µL of the RT reactions, 0.2 µM gene-specific primers, and 1 U rTaq (TaKaRa). A total of 28 to 30 cycles was performed. The primers used in RT-PCR for YAB1 were: YAB1-F (5'-CTTGCTCCTTTTCACCAAGC-3') and YAB1-R (5'-ATGAGCCCAGTTCTTTGCAG-3'). For northern-blotting analysis, 20 µg of total RNA was separated on 1.2% (w/v) denaturing agarose gel before being transferred to nylon membranes. Gene-specific probes were labeled with 32P-dCTP using a Random Primer kit (Invitrogen) and hybridized to the RNA blots. The probes were amplified by PCR using the following primers: GA3ox2-F (5'-CGACCTCTTCCACATCCTCACC-3') and GA3ox2-R (5'-CGTCGGTGGAGACCATCTTGAG-3'); GA2ox3-F (5'-TTCTTCGTCAACGTCGGCGACTCGTTGC-3') and GA2ox3-R (5'-TCTCAAACTGGGCCAGCCTGTTGTCTCC-3'); and GA20ox2-F (5'-GAGACCCTCTCCTTCGGCTTC-3') and GA20ox2-R (5'-CATGGCGGGTAGTAGTTGCAC-3').
The growing shoots with shoot apex of 4-week-old seedlings of YAB1 overexpression transgenic and wild-type rice lines were harvested for quantitative GA analysis. After harvesting, the samples were immediately plunged into liquid N2, freeze-dried, and ground into fine powder using a mill ball. The extraction and purification were carried out as previously described (Coles et al., 1999
Plant materials were fixed in FAA (50% ethanol, 5% acetic acid, 3.7% formaldehyde) overnight at 4°C, dehydrated through a concentration grade of ethanol, cleared through a xylene series, then infiltrated through a series of paraffin, and finally embedded in 100% paraffin melted at 52°C to 54°C. Then 10- to 15-µm-thick microtome sections were mounted on RNase free glass slides. The YAB1 probe was amplified using the gene-specific primers as mentioned above. The PCR fragments were inserted into pBlueScript SK–. The digoxigenin-labeled sense and anti-sense RNA probes were produced by T7 and T3 transcriptase, respectively. The reagents used in the experiments were purchased from Roche.
For the construction of the fusion between the YAB1 promoter and GFP, the GUS sequence in pCAMBIA1381Xb (CAMBIA) was replaced by the GFP gene. The YAB1 promoter was amplified from ‘Nipponbare’ genomic DNA and inserted into the constructed pCAMBIA1381Xb-GFP at the EcoRI and BamHI sites. The primers used in PCR amplification were YAB1prom-F (5'-GGGAATTCTGATCCTGTAGCCCCATCTC-3') and YAB1prom-R (5'-GGGAGCTCCATGCTCCGATGTAAACTGG-3') with an EcoRI adaptor at one end and a BamHI adaptor at the other. Different tissues were harvested at different developmental stages of the transgenic plants, then fixed and sectioned for observation.
Genomic DNA was extracted from young seedlings. A total of 4 µg of DNA was digested with BamHI overnight at 37°C, separated on 1% (w/v) agarose gel, then transferred to a nylon membrane and hybridized according to standard protocols.
DEX (Sigma) was dissolved in ethanol to 30 mM and stored at –20°C before use. Before experiment, DEX was diluted with growth medium to a final concentration of 10 µM. Transgenic siblings from the same lines were divided into three parts: one was treated with DEX, one was treated first with CHX for 1 h then with DEX, and the third received no treatment but an equal volume of ethanol was added to the medium. After 12 h of treatment, the aerial parts of the plants were harvested for RNA extraction, RT-PCR, and quantitative PCR analysis.
Yeast one-hybrid analysis was performed by using a CLONTECH system. The promoter fragment of GA3ox2 was amplified by PCR, which spanned from the initiation ATG codon to –862 and inserted into pHIS2. The primers used were: GA3ox2P-F (5'-GAATTCAGTGGCTGCATGTGAGAATG-3'), GA3ox2P-R (5'-GAGCTCCTGGCAGAAAGCCAGTAACA-3'). The full-length coding sequence of YAB1 was amplified from the full-length cDNA clone using the primer pair YAB1CDS-F (5'-GAATTCGAAATGTCGGTCCAGTTTACATC-3') and YAB1CDS-R (5'- GGATCCTATATCGTCGTCGTCCCTCC-3'). The PCR product was inserted into pGADT7-Rec2 to fuse with the GAL4 activation domain. The constructs were sequenced and used to transform the yeast strain Y187 for one-hybrid analysis.
To produce the YAB1 protein, the full length of the indica YAB1 cDNA was inserted into and translationally fused with the pET-32a expression vector (Novagen) and expressed in Rosetta-gami (DE3) Escherichia coli cells (Novagen). The target protein was purified with B-PER 6xHis Spin Purification kit (Pierce). The 0.8-kb GA3ox2 promoter fragment was cut into two segments by restriction digestion and used as probes in gel shift assays. Oligonucleotides corresponding to the five overlapping regions of the upstream segment of the promoter or to the GARE elements were synthesized and annealed into double strands. The sequence of wild-type GARE oligonucleotide was 5'-TATAACAGATTTATAACAGATT and that of the mutant GARE sites 5'-TATATGAGATTTATATGAGATT. The probes were end-labeled by filling with 32P-dCTP using the Klenow fragment. DNA-binding reactions were performed at room temperature for 20 min in 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 1 mM MgCl2, 5% glycerol, and 50 mg L–1 poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech) and separated on 6% polyacrylamide gels in Tris-glycin (0.3% Tris, 1.88% glycin) buffer.
Transgenic and wild-type seedlings were grown on hydroponic culture media to the four-leaf stage before treatment. Paclobutrazol (at the final concentration of 10 µM) was added to the hydroponic culture 1 d before adding GA3 at the concentrations of 0.01, 0.05, 0.1, 0.5, 1, 10, and 20 µM. The length of the second leaf sheath was measured 10 d after the treatment. The hydroponic culture media and treatments were repeated every 2 d. For YAB1 expression analysis, wild-type seedlings at the four-leaf stage were sprayed with 50 µM GA3 or 10 µM paclobutrazol and harvested at different time points for RNA extraction and quantitative RT-PCR analysis. For expression analysis of GAox genes, transgenic and wild-type plants were sprayed with 50 µM GA3 and harvested at different time points for RNA extraction and quantitative RT-PCR analysis. The expression levels of these genes in these samples were determined relative to time zero, normalized against actin1, and averaged over three biological replicates.
Stems and leaves were harvested from the mature plants of wild type and transgenic ones. Stems were treated with 15% of hydrofluoric acid before dehydration. The procedures of dehydration, clearing, infiltration, and embedding were carried out as mentioned above. The microtome sections (15 µm) were mounted on glass slides for imaging. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AF098752 and BAF12697.
The following materials are available in the online version of this article.
We thank Li Xianhua for technical assistance, Xu Chaiguo for rice field management, Dr. M. Matsuoka for providing slr and gid1 mutants, and Dr. Z. He for sending eui mutants. Received January 25, 2007; accepted March 8, 2007; published March 16, 2007.
1 This work was supported by the National Special Key Program of Rice Functional Genomics, by the National Natural Science Foundation of China, and by the Biotechnology and Biological Sciences Research Council of the United Kingdom (grants to Rothamsted Research).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Dao-Xiu Zhou (dao-xiu.zhou{at}u-psud.fr).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.096586 * Corresponding author; e-mail dao-xiu.zhou{at}u-psud.fr; fax 33–169153424.
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ABSTRACT
RESULTS
-amylase genes (Gubler and Jacobsen, 1992
GR as template. The PCR primers were GR-F (5'-GTCGACAGATCCTGAAGCTCGAAAAAC) with a SalI adaptor and GR-R (5'-GGATCCACCGGCAACAGGATTCAATG) with a BamHI adaptor. The amplified GR fragment was inserted downstream to and in frame with YAB1 in pSport1. The fused YAB1-GR sequence was then cut from pSport1 and inserted into p1301DS digested with KpnI and BamHI. The chimeric protein sequence was (YAB1, amino acids 1–169)-Leu-Ser-Thr-Asp-Pro-(GR, amino acids 507–794). Agrobacterium tumefaciens (strain EHA105)-mediated transformation of rice plants was conducted according to Hiei et al. (1994)