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Keywords:

  • Gibberellin Insensitive Dwarf1;
  • gibberellin;
  • DELLA;
  • receptor;
  • yeast three-hybrid system;
  • quartz crystal microbalance

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In Arabidopsis, three receptors exist for the phytohormone gibberellin. Of the three, only a double loss-of-function mutant (atgid1a atgid1c) shows a dwarf phenotype, while other double and all single mutants show no abnormality in height. In this study we show that the expression of AtGID1b–GUS mRNA, driven by the AtGID1b promoter, is low in inflorescence stems, but may be 10% of AtGID1a–GUS mRNA, driven by the AtGID1a promoter. However, AtGID1bGUS enzymatic activity does not exist in them. This factor strongly suggests that atgid1a atgid1c lacks sufficient AtGID1b protein for normal stem growth. In the stamens of pAtGID1c::AtGID1c–GUS transformants, we detected clear AtGID1cGUS activity, while another atgid1a atgid1b, which has short stamens in its flowers, causes the adhesion of little pollen to stigmas thus leading to its low fertility. We then evaluated the affinity of the AtGID1DELLA interaction by a competitive yeast three-hybrid system and also by QCM apparatus. AtGID1c showed a quite lower affinity to RGL2, the major DELLA protein in floral buds, than AtGID1a or AtGID1b. The low affinity of the AtGID1cRGL2 interaction is likely to be responsible for the failure of AtGID1c to hold RGL2, which is required for normal stamen development. Taken together with expressional information of DELLA genes, we propose that in a double loss-of-function mutant of gibberellin receptors, the emergence of any phenotype(s) depends on the abundance of the remaining receptor and its preference to DELLA proteins existing at a target site.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The phytohormone, gibberellin (GA) controls important physiological phenomena in plants, such as seed germination, stem elongation, and formation of the floral bud. After the identification of a GA receptor, Gibberellin Insensitive Dwarf1 (GID1) in rice, and three orthologs in Arabidopsis (Ueguchi-Tanaka et al., 2005; Nakajima et al., 2006), the transduction of the GA signal was clearly explained; namely, (i) key protein(s) called ‘DELLA’ interfere with the transduction of the GA signal usually when little GA exists, (ii) once GA is perceived by GID1, the GA–GID1 complex has an affinity to DELLA, (iii) DELLA is captured by the GID1–GA complex and then inactivated (Ariizumi et al., 2008; Ueguchi-Tanaka et al., 2008), and (iv) DELLA is diminished by proteasomes through ubiquitination (McGinnis et al., 2003; Dill et al., 2004; Fu et al., 2004; Gomi et al., 2004). As a result, the weakening of DELLA’s function initiates the GA-triggered actions (Fleet and Sun, 2005).

In Arabidopsis, three GID1s and five DELLAs exist. Therefore, a more complicated regulation of GA signaling is expected in this plant because only one GID1 and one DELLA function in rice. Analyses of multiple loss-of-function mutants for GID1s in Arabidopsis strongly support this idea, namely that any single knock-out mutant of GID1s did not show any visible phenotype, which implies that either or both of the remaining GID1(s) function sufficiently. However, only the atgid1a atgid1c double mutant shows a dwarf phenotype. This finding suggests that AtGID1b does not function effectively in stem elongation. The atgid1a atgid1b atgid1c triple mutant seeds never germinated voluntarily, and after removing their testae with forceps, the embryos grew but were much shorter than the atgid1a atgid1c double mutant (Griffiths et al., 2006; Iuchi et al., 2007; Willige et al., 2007). Thus, AtGID1b functions poorly in the stem. Another double loss-of-function mutant atgid1a atgid1b has short stamens, which leads to its low fertility (Griffiths et al., 2006; Iuchi et al., 2007; Willige et al., 2007). Therefore, it supposed that AtGID1c probably functions less effectively than AtGID1a or AtGID1b in the floral organ. Multiple loss-of-function mutants for DELLAs have also been reported (Dill and Sun, 2001; King et al., 2001; Fu and Harberd, 2003; Cheng et al., 2004; Tyler et al., 2004; Yu et al., 2004). Particularly in stem elongation, it has been reported that functional losses of RGA and GAI lead to a clear slender phenotype, but not to any other clearer effects, suggesting that other DELLAs might have decisive functions there (Dill and Sun, 2001; King et al., 2001; Fu and Harberd, 2003). In floral development, it reported that RGA, RGL1, and RGL2 act as dominant DELLAs (Cheng et al., 2004; Tyler et al., 2004; Yu et al., 2004). Taken together, many functional overlaps among three GID1s or five DELLAs exist, although their specific roles are likely to exist at a specific site.

So in this report, we tried to dissect the molecular mechanisms required for the phenotypic emergence of the gid1 double mutants by two approaches. Firstly we analysed the amounts of GID1 mRNA and of its gene product in the pGID1::GID1-β-glucuronidase (GUS) transformants to detect a quantitative suspension of GA-signal transduction by insufficiency of the receptor gene or its product. Secondly we evaluated the affinity value for the binding between a GID1 and a DELLA protein for detection of qualitative suspension of GA-signal transduction by the dissociation of the binding with its low affinity.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Quantification of AtGID1 and DELLA mRNAs in gid1 double mutants

Firstly we quantified the absolute amount of AtGID1 transcripts by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), for fear that the phenotypes of double mutants would simply be caused by a decrease of the remaining receptor gene. We prepared total RNA from double mutants and parental WTs. As shown in Figure 1(a), in atgid1a atgid1c inflorescence stems, the amount of AtGID1b transcript did not show a big change compared with that of WT. Also in atgid1a atgid1b floral buds, the amount of AtGID1c transcript was not that different with that of WT (Figure 1c). In addition to AtGID1 transcripts, we also quantified the absolute amount of DELLA transcripts (Figure 1b,d). Generally, no large change in their amounts was detected, but the amount of RGL1 transcript was lower in both atgid1a atgid1c inflorescence stems and atgid1a atgid1b floral buds compared with that in WT. Additionally, although we could not detect any phenotypic abnormalities in atgid1a atgid1c floral buds, an increase of AtGID1b transcript and a decrease of RGL1 transcript were detected (Figure 1c,d).

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Figure 1.  Quantification of AtGID1 and DELLA mRNAs. Upper panels: absolute quantification of each AtGID1 transcript (a) and of each DELLA transcript (b) in 10 inflorescence stems. Lower panels: absolute quantification of each AtGID1 transcript (c) and of each DELLA transcript (d) in 10 floral buds. Col, Columbia; Nos, Nossen; N/C, Nos/Col hybrid; abC, atgid1a atgid1b; aBc, atgid1a atgid1c; and Abc, atgid1b atgid1c. All transcripts were quantified by qRT-PCR based on the Act2 gene. N, not detected. We repeated the quantification independently twice and obtained similar results.

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To better assess the transcriptional level of those genes at a much higher resolution, we decided to quantify both AtGID1 and DELLA transcripts by using qRT-PCR only in filaments. We removed all anthers from stamens of just blooming WT (Columbia) flowers, and collected ca. 300 filaments twice. These results are shown in Figure S1. In WT filaments, AtGID1a and AtGID1b transcripts were comparable with each other, and RGL1 and RGL2 transcripts were dominant in five DELLA genes.

Spatial expression pattern of AtGID1 mRNAs

Next, we prepared transformants carrying a β-glucuronidase (GUS) gene driven by an AtGID1 promoter (Figure 2a). The GUS gene was fused with the C-terminus of each genomic AtGID1 coding region, including one intron. We obtained eight lines (p1a::1a) for pAtGID1a::AtGID1a–GUS, two lines (p1b::1b) for pAtGID1b::AtGID1b–GUS, and four lines (p1c::1c) for pAtGID1c::AtGID1c–GUS. We firstly focused on inflorescence stems of the transformants. Almost no GUS activity was detected at the p1b::1b site, while clear GUS activity was detected at both p1a::1a and p1c::1c sites (Figure 2b). Then we absolutely quantified the AtGID1–GUS transcript in inflorescence stems of each line with specific probes for the GUS gene; the AtGID1b–GUS transcript surely existed in inflorescence stems of p1b::1b (Figure 2c). Next, we focused on transformants’ floral buds. We detected clear GUS activity in the stamens of all lines examined, even in p1c::1c (Figure 2b).

image

Figure 2.  Analysis of pAtGID1::AtGID1–GUS transformants. (a) pAtGID1::AtGID1–GUS constructs. (b) Top part (i–iii) and growing zone (iv–vi) of an inflorescence stem, and a flower (vii–ix) of each transformant. We independently observed a few lines of each, and obtained similar staining patterns. Bars = 1 mm. (c) Absolute quantification of GUS transcript: (i) Growing zone of five inflorescence stems of each transformant; and (ii) five floral buds of each transformant. We repeated the quantification independently twice and obtained similar results.

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Competitive interaction of two AtGID1s with one DELLA in yeast

We had already established a yeast two-hybrid (Y2H) system that showed a GA-dependent interaction between an AtGID1 and a DELLA (Nakajima et al., 2006). Then, to evaluate the affinity for the interaction of AtGID1–DELLA in vivo, we established a yeast three-hybrid (Y3H) system that contains three molecules in yeast using a GID1 as bait (BDGID1), a DELLA as prey (ADDELLA), and another GID1 (2ndGID1) as a competitor to BDGID1. In this situation, we can easily compare the interaction affinity of ADDELLA to BDGID1 with that to 2ndGID1 by the growth of yeasts. In other words, the yeasts grow when the interaction of BDGID1–ADDELLA is stronger than that of 2ndGID1–ADDELLA (Figure 3a). Contrarily, when the interaction of BDGID1–ADDELLA is equivalent to or weaker than that of 2ndGID1–ADDELLA, the yeasts can not grow as 2ndGID1 removes ADDELLA from BDGID1 (Figure 3b). To verify the system, we examined control experiments: (i) we constructed a set of vectors in which nothing was ligated to the MCS II site of pBridge vector for 2ndGID1. All control yeasts grew on selection medium in the presence of GA (Figure S2a), similarly to the Y2H results (Nakajima et al., 2006); and (ii) we confirmed that all yeasts carrying a set of BDGID1–ADDELLA–2ndGID1 grew on non-selection medium without GA (Figure S2b).

image

Figure 3.  Competition of two AtGID1s to interact with a DELLA in yeast. (a) Y3H system consists of three molecules; one AtGID1 as bait (BDGID1), a DELLA as prey (ADDELLA), and another AtGID1 (2ndGID1) as a competitor. On selection medium, yeasts can grow when the BDGID1–ADDELLA interaction is stronger than that of 2ndGID1–ADDELLA. (b) In contrast, when the BDGID1–ADDELLA interaction is equivalent to or weaker than that of 2ndGID1–ADDELLA, yeasts can not grow because 2ndGID1 removes ADDELLA from BDGID1. (c) Y3H assay (30°C, 7 days) on selection medium in the presence of GA4 (10 μm) and 3-AT (10–100 mm). We examined the assay four times independently and obtained similar results each time.

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Results are shown in Figure 3(c). We judged the survival of yeasts in the presence of 3-AT (10–100 mm). Interestingly, when RGL2 was used as prey, all yeasts carrying BDAtGID1c were difficult to grow, regardless of whether 2ndAtGID1a or 2ndAtGID1b were adopted as a competitor. Contrarily, all yeasts carrying 2ndAtGID1c grew, regardless of whether we adopted BDAtGID1a or BDAtGID1b as a competitor. Taken together, it is understood that the interaction of AtGID1c–RGL2 was fully blocked because both AtGID1a and AtGID1b could preferentially form a complex with RGL2, while the AtGID1a–RGL2 or AtGID1b–RGL2 interaction was maintained, even in the presence of AtGID1c. RGL2 showed the lowest preference to AtGID1c among the three GID1s. In addition, RGL2 showed the highest preference to AtGID1a because the yeast carrying a set of BDAtGID1a–ADRGL2–2ndAtGID1b grew while the yeast carrying a set of BDAtGID1b–ADRGL2–2ndAtGID1a could not. Both RGL1 and RGL3, when used as prey, showed the lowest preference to AtGID1b among the three because yeast carrying a set of BDAtGID1a/c–ADRGL1/3–2ndAtGID1b grew while yeast carrying a set of BDAtGID1b–ADRGL1/3–2ndAtGID1a/c could not. Unlike the RGLs, when GAI or RGA were used as prey, both appeared to show a higher preference to AtGID1b, because the yeast carrying a set of BDAtGID1b–ADGAI/RGA–2ndAtGID1a/c grew, while yeast carrying a set of BDAtGID1a/c–ADGAI/RGA–2ndAtGID1b could not. By western blotting, we successfully detected comparable amounts of both BDGID1 and 2ndGID1 proteins from all yeasts grown on selection medium (Figure S3). This situation explains the yeasts can grow because 2ndGID1 is powerless to remove ADDELLA from BDGID1 but not because the amount of 2ndGID1 is too little to function in the yeast. Here, it was clearly shown that some advantageous combinations exist for the AtGID1–DELLA interaction in yeasts.

Direct evaluation for the AtGID1–DELLA interaction by quartz crystal microbalance

To verify the preferences of the AtGID1–DELLA interaction more directly, we analysed the interaction kinetics of both molecules in vitro by using a quartz crystal microbalance (QCM). The weight of the gold electrode film on the quartz crystal and the change in its resonance frequency are strongly related. Therefore, the QCM can detect a tiny weight change on the gold electrode where some molecules bind to the molecules on the electrode surface (Sauerbrey, 1959; Sato et al., 2004). A purified recombinant glutathione-S-transferase-fused DELLA (GSTDELLA) was immobilized onto the surface of a sensor chip. We measured the oscillation frequency of the quartz crystal on the chip when various concentrations of a purified recombinant thioredoxin-fused GID1 (TrxGID1) were added in the presence of GA4 (10 μm).

As we had detected in Y3H results that RGL2 showed the lowest preference to AtGID1c among the three GID1s, we decided to focus on the AtGID1c–RGL2 combination (Figure 3c). The dissociation constant value (Kd) was calculated based on Scatchard plots obtained from sensorgrams (Figure 4). The Kd value for the TrxAtGID1c–GSTRGL2 interaction was calculated to be 1.0 × 10−7 m, which was one-tenth the affinity of the TrxAtGID1a–GSTRGL2 interaction (Kd = 1.3 × 10−8 m). We could not perform the experiments with TrxAtGID1b because of its low amount in Escherichia coli. As a reference, we also employed RGA, which showed preference to AtGID1a, comparable with preference to AtGID1c, in the Y3H assay (Figure 3c). As expected, the value for the TrxAtGID1a–GSTRGA interaction (Kd = 5.1 × 10−8 m) was very similar to that for the TrxAtGID1c–GSTRGA interaction (Kd = 8.3 × 10−8 m). Taken together from the AtGID1–DELLA combinations examined here, it is clear that their affinity values obtained from QCM and the preferences from Y3H show a very similar tendency to each other.

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Figure 4.  Scatchard plots for the AtGID1–DELLA interaction. QCM experiments were performed at 25°C by using both purified recombinant Trx–AtGID1 (TrxAtGID1) and GST–DELLA (GSTDELLA). GA4 was added to the reaction mixture at a final concentration of 10 μm. (a) TrxAtGID1a–GSTRGL2, (b) TrxAtGID1c–GSTRGL2, (c) TrxAtGID1a–GSTRGA, and (d) TrxAtGID1c–GSTRGA. The Kd value were calculated as (−1) × (slope)−1. This experiment was repeated at least twice and similar values were obtained in all combinations.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In this report, to elucidate the reasons why a dwarf phenotype emerged in only atgid1a atgid1c double mutants and why abnormally short stamens emerged in only atgid1a atgid1b double mutants, we adopted two standpoints: (i) comparison of spatial expression patterns of AtGID1 genes by preparation of AtGID1-fused GUS transformants driven by an AtGID1 promoter; and (ii) evaluation of the affinity for the AtGID1–DELLA interaction.

We first assessed the dwarfism shown in atgid1a atgid1c double mutants. In inflorescence stems of p1b::1b transformants, we could not detect any staining for AtGID1b-fused GUS protein (Figure 2b). There was no possibility that AtGID1b–GUS had insufficient enzymatic activity to hydrolyze substrates, because clear staining was detected at other sites (Figure 2b). Then we quantified AtGID1bGUS mRNA in p1b::1b transformants, and detected some amount of AtGID1bGUS mRNA in their inflorescence stems (Figure 2c). This finding implies that the low level of GUS staining in the p1b::1b inflorescence stems was caused by a low amount of AtGID1b–GUS product. In other words, the low GUS staining in the p1b::1b inflorescence stems may be caused by the low stability of AtGID1b protein. Namely, a dwarf phenotype in only atgid1a atgid1c double mutants can simply be explained by the lack of a remaining GA-receptor AtGID1b in inflorescence stems. An unstable mechanism for AtGID1b is unknown, however the unique characteristic of AtGID1b in inflorescence stems may be advantageous to plants, for example because it prevents plants from becoming slender. In the atgid1a atgid1c floral buds, the amount of RGL1 transcript decreased (Figure 1d) and that of AtGID1b increased (Figure 1c), although we could not observe any phenotypic abnormality in them. These transcriptional changes suggest that both inflorescence stems and floral buds were nearly starved of a GA signal in atgid1a atgid1c double mutant, or that an unknown signal might be transported to floral buds from inflorescence stems.

Similar to the above-mentioned discussion on the atgid1a atgid1c dwarfism, we focused on the GUS-staining result in flowers of p1c::1c transformants to clarify why the stamens of atgid1a atgid1b double mutants were shorter than those of other double mutants. However, unlike the case of atgid1a atgid1c dwarfism, we detected clear GUS staining in stamens of p1c::1c transformants (Figure 2b), and we also detected high expression of the AtGID1c–GUS gene in floral buds (Figure 2c). Therefore, AtGID1c may be stable there, while the expression of AtGID1c–GUS mRNA might be artificially exaggerated probably because we had omitted the 3′-untranslated region (3′-UTR) of genomic AtGID1c DNA when fused to the GUS gene. Strangely, this exaggeration of AtGID1c–GUS expression did not reflect the GUS staining intensity shown in p1c::1c flowers (Figure 2b). Therefore AtGID1c may be moderately stable in flowers because of a combination of high AtGID1c expression and not-so-stable AtGID1c protein. We are however, unable to explain the abnormal stamen length of the atgid1a atgid1b double mutant by the absence of a remaining receptor AtGID1c in it.

We then shifted our focus to another possibility: AtGID1c exists but it can not function effectively in stamens. We focused on the affinity of the AtGID1–DELLA interaction in the presence of GA. In our previous report (Nakajima et al., 2006), we had already recognized that the interaction between an AtGID1 and a DELLA happens in all 15 combinations by using the Y2H system. Based on that system, we developed a Y3H system fitted for the evaluation of an affinity between an AtGID1 and a DELLA. In addition to the in vivo system, the QCM added more direct results by evaluating the affinity values (Figure 4). As summarized in Figure 5, five DELLAs showed a variety of preferences to AtGID1s, i.e., GAI and RGA showed the highest preference to AtGID1b, RGL2 preferred AtGID1a, while no DELLA preferred AtGID1c. Thus, although DELLAs do not always show a high preference to AtGID1b, AtGID1b has a higher affinity (Kd = 4.8 × 10−7 m) to the most active gibberellin GA4 than other GID1s have (Kd = ca. 2 × 10−6 m), as shown in our previous report (Nakajima et al., 2006). This factor implies that an affinity for the GID1–DELLA interaction does not depend greatly on the affinity for the GID1–GA interaction. In contrast, RGL1 and RGL3 showed the lowest preference to AtGID1b, GAI to AtGID1a, and RGL2 to AtGID1c. As mentioned in the ‘introduction’, the following incidents are thought to be required for initiating GA-triggered action in plants: (i) the GA receptor captures a DELLA after the perception of GA; (ii) the receptor–GA complex continues to hold a DELLA to inactivate the DELLA; and (iii) the total amount of active DELLAs decreases under a threshold which is not enough to suppress gene expression related to the GA-triggered action. Therefore, in point (ii), we can envisage that the lower affinity the interaction between an AtGID1 and a DELLA shows, the greater the difficulty AtGID1 has in continuing to hold the DELLA tightly with the risk of letting the DELLA loose. In Figure 5, the combinations of AtGID1b–RGL1/3, AtGID1a–GAI, and AtGID1c–RGL2, show low affinity to form a complex of the two molecules. These combinations constitute the risky cases for the dissociation of the complex in point (ii).

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Figure 5.  Schematic illustration of the preference of the AtGID1–DELLA interaction. In the AtGID1–DELLA combination a higher affinity exists than in other combinations. The stronger combination is linked by a bold arrow, while the weaker combination is linked by a narrow, dotted arrow.

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The discussion now turns to the short stamens that emerged in atgid1a atgid1b double mutants. The remaining receptor AtGID1c shows a low affinity to RGL2 (Figure 5). And as shown in Figure 1(d), the RGL2 transcript was one of the dominant DELLA mRNAs in floral buds of atgid1a atgid1b. Additionally, the RGL2 transcript was also detected as a major DELLA in filaments of WT flowers (Figure S1). Thus, a high amount of RGL2 should lead to the failure of AtGID1c to continue capturing RGL2 fully, which should interrupt the transduction of the GA signal and be the main reason for the abnormality of the stamens. Here, questions remain: why do the stamens of atgid1a atgid1c not have any phenotypes, even though they should be at a disadvantage with the AtGID1b–RGL1 or AtGID1b–RGL3 combination? And why do the stamens of atgid1b atgid1c also not have any phenotypes, even though they should be at a disadvantage with the AtGID1a–GAI combination? The former question can be explained by the low expression of RGL3 mRNA (Figure 1d and Figure S1), and by the decrease of RGL1 mRNA compared with the transcript in WT (Figure 1d). The latter question may be answered by the fact that the basal expression level of AtGID1a is high in general (Figure 1c and Figure S1). To further validate these explanations, the quantification of AtGID1/DELLA proteins in double mutants needs to be more conclusive, although this will not be easy since they exist in low amounts.

In this report, a combination of the Y3H experiment and QCM analysis provided new insight into the relation between the preference of the GID1–GA interaction and that of the GID1–DELLA interaction. Difference in the preference for the GID1–DELLA interaction was conclusive. Moreover, preference of the GID1–DELLA interaction can divide the five Arabidopsis DELLAs into two groups: one is GAI and RGA which show the highest preference to AtGID1b among the three GID1s, while the other is RGLs, which show the highest preference to AtGID1a. The two groups are separated by a gap on a phylogenic tree drawn with full-length DELLA sequences (Hirano et al., 2007). We assume that AtGID1b is an evolutional form developed from AtGID1a or AtGID1c (Nakajima et al., 2006; Hirano et al., 2007), because of its higher affinity to GA (Nakajima et al., 2006), and also because the prototype combinations for GA signaling in Arabidopsis may be AtGID1a/c and GAI/RGA. If so, AtGID1b is thought to be required for Arabidopsis to transmit the signal to GAI/RGA more effectively than AtGID1a/c does, because both GAI and RGA are major DELLAs in inflorescence stems and show higher affinity to AtGID1b than to AtGID1a/c. Recently, Ariizumi et al. (2008) reported that in the sly1 background, in which the function of an F-box subunit SLY1, a member of E3 ubiquitin ligase complex for GA signaling, is weakened or almost lost, and that the AtGID1b over-expression (OE) lines have much longer inflorescence stems than other AtGID1-OE lines. In this situation, the higher affinity of AtGID1b to GA (Nakajima et al., 2006) does not seem to depend on the phenotypic character of AtGID1b-OE lines, because the application of GA to all AtGID1-OE lines showed almost no effect and endogenous GA should not be a limiting factor for elongation of their inflorescence stems (Ariizumi et al., 2008). The data of their OE lines may be consistent with our above-mentioned assumption that AtGID1b is more effective to inactivate GAI or RGA than AtGID1a/c, although we can not yet explain why the AtGID1b proteins can fully exist and function in inflorescence stems of gene-OE lines in a sly1 background. The reason for these preferences will be the focus of our future research.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

gid1 double loss-of-function mutant lines

We used three mutants (atgid1a-Ds atgid1b-Ds, atgid1a-SALK atgid1c-1, and atgid1b-Ds atgid1c-1) in this experiment. The atgid1a-1 (Ds13-1770-1), atgid1a-2 (SALK_044317), and atgid1b-1 (Ds11-3970-1) shown in our previous report (Iuchi et al., 2007) were renamed as atgid1a-Ds, atgid1a-SALK, and atgid1b-Ds, respectively.

qRT-PCR

Total RNA was isolated by using Plant RNA Isolation reagent (Invitrogen, Carlsbad, CA) and RT reaction was setup using M-MLV Reverse Transcriptase (Invitrogen) following the manufacturer’s instructions. The vectors for standard curves were constructed by inserting the coding sequences into the pGEM T-easy vector (Promega). The primers used are the same as those for constructions of vectors for the Y3H assay (for pBridge MCS I and for pGADT7 MCS in Table S1). The amount of RNA of each gene in each cDNA sample was assessed by qRT-PCR using a standard curve derived from the vector, including the corresponding gene. The Real-Time PCR instrument (Thermal Cycler Dice), reagent (SYBR Premix Ex Taq) and primer sets were obtained from Takara Bio Inc. (Shiga, Japan) and executed according to the manufacturer’s instructions. The primer sequences are listed in Table S1. A standard curve from the Act2 gene was used to ensure the total amount of RNA from samples.

GUS assay for pAtGID1::AtGID1–GUS transformants

The genomic DNA fragments, including 3 kbp upstream and the coding region of AtGID1s, were cloned into the Hind III/BamH I sites of pBI121 GUS expression binary vector (Clontech). The resulting vectors were introduced into Agrobacterium GV3010. Arabidopsis was transformed by the floral dip method (Clough and Bent, 1998). For the GUS enzymatic assay, tissue was pre-fixed in ice-cold 90% (v/v) acetone for 20 min on ice, rinsed with 100 mm sodium phosphate buffer (pH 6.9), vacuum infiltrated for 20 min with staining solution (100 mm sodium phosphate buffer (pH 6.9), 10 mm EDTA, 0.1% (v/v) Triton X-100, 10 mm potassium ferricyanide, 10 mm potassium ferrocyanide, 0.5 mg ml−1 X-gluc) and incubated at 37°C for 14 h. Samples were incubated in 70% (v/v) ethanol and then decolorizing solution (ethanol:acetic acid = 6:1 (v/v)) for 2 h. Decolorized samples were viewed under a stereoscopic microscope (SZX10, Olympus).

Y3H assay

Saccharamyces cerevisiae strains AH109 and Y187, plasmids pGADT7 and pBridge were from Takara Bio Inc. pBridge was restricted with a pair of adequate restriction enzymes shown in Table S1, and used for the expression of a GID1 fused with a GAL4-BD domain (BDGID1) and another GID1 (2ndGID1). Each GID1 gene for BDGID1 was excised from the pGBKT7–GID1 plasmid already obtained for Y2H (Nakajima et al., 2006) using EcoR I and Pst I. Their fragments were ligated into pBridge MCS I for BDGID1 and into MCS II for 2ndGID1. In-frame insertion and correctness of the sequence was verified by DNA sequencing (ABI PRISM 310 Genetic Analyzer, Applied Biosystems Inc, CA). Strain Y187 was transformed with the pBridge vector carrying BDGID1 and 2ndGID1 genes or only a BDGID1 gene, and selected on SD media without l-tryptophan and uracil. Strain AH109 carrying a plasmid pGADT7–DELLA, which was obtained previously (Nakajima et al., 2006), grew on SD media without l-leucine, uracil, and l-methionine. By mating both strains, the transformants carrying both plasmids of pBridge and pGADT7–DELLA were selected on SD medium without l-leucine, l-tryptophan, uracil, and l-methionine. For the assay, transformants were incubated on SD media without adenine hemisulfate, l-histidine, l-leucine, l-tryptophan, uracil, and l-methionine but in the presence of GA4 and 3-aminotriazole (3-AT).

Immunoblotting

Yeasts (100 mg) were lysed with 0.5 ml of Y-PER® Yeast Protein Extraction Reagent (Takara Bio Inc.) containing a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and Benzonase® Nuclease (Merck & Co., Inc., Whitehouse Station, NJ), according to manufacturer’s instruction. Soluble proteins were subjected to chloroform–methanol precipitation and subsequently separated on SDS-PAGE (10%). Detection of BDGID1 and 2ndGID1 were performed with polyclonal anti-GID1 antiserum generated against amino acids 34–47 and 243–256 of GID1a and SuperSignal® West Dura Extended Duration Substrate (Takara Bio Inc.).

QCM

Preparation of recombinant proteins was according to an expression system with E. coli described previously (Nakajima et al., 2006) with specific primers (Table S1). Purified GSTDELLA (0.3 mg ml−1 in 50 mm Tris–HCl, pH 8.0) was immobilized onto a QCM sensor chip (Initium, Tokyo, Japan) as described in the manufacturer’s protocol. After washing with distilled water, the sensor chip was set to AFFINIX Q (Initium), and then soaked in 8 ml of 20 mm Tris–HCl, 100 mm NaCl (pH 7.6) in the presence of GA4 (10 μm). Purified TrxGID1 (0.5 mg ml−1 in 50 mm phosphate buffer, pH 7.0) was added stepwise to the solution, and the frequency change for QCM was monitored at 25°C. Kinetics for the interaction was analysed with AQUA (Initium) software.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by grants to M.N., M.M. from the Ministry of Education, Culture, Sports, Science and Technology of Japan, to M.N. from the Naito Foundation, and to M.N., T.A. from the Bio-oriented Technology Research Advancement Institution (PRO-BRAIN).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Absolute quantification of AtGID1 (a) and DELLA (b) transcripts in ca. 300 filaments. All transcripts were absolutely quantified by qRT-PCR based on the Act2 gene. Quantification was independently repeated twice, and similar results were obtained.

Figure S2. Control experiments for competitive Y3H assay. (a), The assay for the combination of one AtGID1 as bait (BDGID1) and a DELLA protein as prey (ADDELLA) was examined on selection medium in the presence of GA4 (10 μm) and 3-AT (10-100 mM). Nothing ligated to the pMet site (2ndvec). (b) On the non-selection medium (SD without L-leucine, L-tryptophan, uracil, and L-methionine) in the absence of GA4, the assay for all BDGID1-ADDELLA-2ndGID1 combinations was examined. 3-AT (100 mm) was added. Both assays were repeated three times independently, and similar results were obtained each time.

Figure S3. Immunological detection of two GID1s in all surviving yeasts for three-hybrid assay. All BDGID1s have a molecular size of around 57 kDa (arrow head). All 2ndGID1s have a molecular size of around 40 kDa (arrow). Asterisks (*) indicate bands that reacted non-specifically with anti-GID1 antiserum.

Table S1. Sequences for primers.

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