Trails to the gibberellin receptor, GIBBERELLIN INSENSITIVE DWARF1.

The researches on the identification of gibberellin receptor are reviewed from the early attempts in 1960s to the identification of GIBBERELLIN INSENSITIVE DWARF1 (GID1) as the receptor in 2005. Unpublished data of the gibberellin-binding protein in the seedlings of adzuki bean (Vigna angularis) are also included, suggesting that the active principle of the gibberellin-binding protein was a GID1 homolog.

The rice FISH BONE gene encodes a tryptophan aminotransferase, which affects pleiotropic auxin-related processes.

Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole-3-pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole-3-acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin-related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.

A highly sensitive, quick and simple quantification method for nicotianamine and 2'-deoxymugineic acid from minimum samples using LC/ESI-TOF-MS achieves functional analysis of these components in plants.

A highly sensitive quantitative method for assaying nicotianamine (NA) and 2'-deoxymugineic acid (DMA) using liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) was developed. The amino and hydroxyl groups of NA and DMA were derivatized using 9-fluorenylmethoxycarbonyl chloride. The amounts of NA and DMA in 10 mul of xylem sap from rice cultivated under iron (Fe)-sufficient and Fe-deficient conditions were quantified without concentration. In Fe-sufficient plants, the concentrations of NA and DMA were almost equal to that of Fe. In Fe-deficient plants, the concentration of NA did not change significantly, whereas that of DMA increased markedly.

Differential expression and affinities of Arabidopsis gibberellin receptors can explain variation in phenotypes of multiple knock-out mutants.

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, AtGID1b-GUS 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 AtGID1c-GUS 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 AtGID1-DELLA 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 AtGID1c-RGL2 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.

A new gibberellin detection system in living cells based on antibody V(H)/V(L) interaction.

As a new detection method of bioactive gibberellin A(4) (GA(4)) in living cells, a combined system of GA(4)-dependent interaction of V(H) and V(L) composed of a variable region fragment (Fv) of anti-GA(4) antibodies and protein-fragment complementation assay (PCA) was developed. First, when V(H) and V(L) were displayed in proximity on a phage, they could constitute a functional Fv. Thereafter, V(H) and V(L) were shown to interact with each other in a GA(4)-dependent manner. We then applied this interaction to PCA using GFP as a reporter. V(H) fused to the C-terminal half of GFP and V(L) fused to the N-terminal half of GFP were simultaneously expressed in Escherichia coli. The E. coli in which these fusion proteins were inductively produced in the presence of GA(4) showed clear GFP fluorescence, while those in the absence of GA(4) showed only scarce GFP fluorescence, demonstrating the feasibility of this system to detect GA(4) in living organisms.

Immunomodulation of gibberellin biosynthesis using an anti-precursor gibberellin antibody confers gibberellin-deficient phenotypes.

Immunomodulation is a means to modulate an organism's function by antibody production to capture either endogenous or exogenous antigens. We have recently succeeded in obtaining gibberellin (GA)-deficient phenotypes in Arabidopsis thaliana by using anti-bioactive GA antibodies. In this study, a single-chain antibody (scFv) against GA(24), a precursor GA, was utilized to repress the biosynthesis of bioactive gibberellins. Stable accumulation of the scFv in endoplasmic reticulum (ER) was achieved by being produced as a fusion with GFP as well as KDEL ER-retention signal. The transgenic plants showed GFP fluorescence in the reticulate cortical ER network in epidermal cells. The GFP-scFv fusion produced in plants maintained its binding activity. The transgenic plants showed GA-deficient phenotypes, including reduced rosette leaf development, delayed flower induction and reduced stem elongation of the main culm, especially in the early stage of inflorescence growth. Contrarily, stem elongation of the main culm at a later stage, or that of lateral shoots was much less affected by scFv production. These phenotypes were different from anti-bioactive GA scFv-producing lines, whose stem elongation was continuously repressed throughout the inflorescence development. The GA-deficient phenotypes were recovered by treatment with GA(24) and bioactive GA(4), the latter being more effective. The transgenic lines contained conspicuously higher endogenous GA(24) and clearly less GA(4) than wild-type plants. The expression of GA 20-oxidase and GA 3-oxidase genes, which are feedback-regulated by GA signaling, were up-regulated in those plants. These results demonstrate that the scFv trapped GA(24) in ER and inhibited metabolism of GA(24) to bioactive GA(4).

Immunomodulation of bioactive gibberellin confers gibberellin-deficient phenotypes in plants.

Immunomodulation is a means to modulate an organism's function by antibody production to capture either endogenous or exogenous antigens. This method was applied to plants to repress the function of gibberellins (GAs), a class of phytohormones responsible for plant elongation, by anti-bioactive GA antibodies. Two different antibodies were produced in Arabidopsis as single-chain variable fragment (scFv) fused to green fluorescent protein (GFP) with four different subcellular localizations: endoplasmic reticulum (ER), cytosol, apoplastic space or the outer surface of the plasma membrane. When targeting scFv-GFP to ER, plants showed the highest accumulation of scFv-GFP, with binding activity, strong GFP fluorescence in ER-derived compartments and mild but clear GA-deficient phenotypes, including a smaller leaf size, delayed bolting, shorter inflorescence length and decreased germination. Plants expressing scFv-GFP in ER responded to exogenous GA(4) and contained 15-40 times greater endogenous GA(4) than wild-type plants. They also showed increased gene expression for GA3ox1, GA20ox1 and GA20ox2, but decreased expression for GA2ox1, which are feedback and feedforward regulated by GA signalling, respectively. These results suggest that the level of free functional GA(4) decreased when trapped in the ER with scFv to the extent that mild GA-deficient phenotypes were created. A dramatic increase in the total sum of GA(4) (free plus scFv-GFP bound) was detected as a result of the up-regulation of GA biosynthesis (feedback regulated), and a decrease in GA(4) catabolism as a result of protection by scFv-GFP binding. This study demonstrates that the use of immunomodulation to inhibit the action of bioactive GAs is an effective method of creating GA-deficient plants.

Defense-related signaling by interaction of arabinogalactan proteins and beta-glucosyl Yariv reagent inhibits gibberellin signaling in barley aleurone cells.

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins present at the plasma membrane and in extracellular spaces. A synthetic chemical, beta-glucosyl Yariv reagent (beta-GlcY), binds specifically to AGPs. We previously reported that gibberellin signaling is specifically inhibited by beta-GlcY treatment in barley aleurone protoplasts. In the present study, we found that beta-GlcY also inhibited gibberellin-induced programmed cell death (PCD) in aleurone cells. We examined the universality and specificity of the inhibitory effect of beta-GlcY on gibberellin signaling using microarray analysis and found that beta-GlcY was largely effective in repressing gibberellin-induced gene expression. In addition, >100 genes were up-regulated by beta-GlcY in a gibberellin-independent manner, and many of these were categorized as defense-related genes. Defense signaling triggered by several defense system inducers such as jasmonic acid and a chitin elicitor could inhibit gibberellin-inducible events such as alpha-amylase secretion, PCD and expression of some gibberellin-inducible genes in aleurone cells. Furthermore, beta-GlcY repressed the gibberellin-inducible Ca2+-ATPase gene which is important for gibberellin-dependent gene expression, and induced known repressors of gibberellin signaling, two WRKY genes and a NAK kinase gene. These effects of beta-GlcY were also phenocopied by the chitin elicitor and/or jasmonic acid. These results indicate that gibberellin signaling is under the regulation of defense-related signaling in aleurone cells. It is also probable that AGPs are involved in the perception of stimuli causing defense responses.

Identification and characterization of an Ipomoea nil glucosyltransferase which metabolizes some phytohormones.

A glucosyltransferase gene InGTase1 was identified from the immature seeds of morning glory (Ipomoea nil), whose product shows a broad substrate-preference, including that of some phytohormones. When 2-trans-abscisic acid, indole-3-acetic acid, salicylic acid (SA) or (+/-)-jasmonic acid was reacted with InGTase1 and UDP-[(14)C]-glucose, each (14)C-labeled compound with high polarity was detected after thin layer chromatography. SA metabolites were identified as SA glucosyl ester by using (1)H NMR and GC/MS. Detailed substrate-preferences of InGTase1 were examined with some analogous compounds, which elucidated that the arm length and/or orientation of a carboxyl group of the compounds or its surrounding electron density severely affected the enzymatic activity. The broad substrate-preference will greatly contribute to the synthesis of various glucoconjugates.

Molecular interactions of a soluble gibberellin receptor, GID1, with a rice DELLA protein, SLR1, and gibberellin.

GIBBERELLIN INSENSITIVE DWARF1 (GID1) encodes a soluble gibberellin (GA) receptor that shares sequence similarity with a hormone-sensitive lipase (HSL). Previously, a yeast two-hybrid (Y2H) assay revealed that the GID1-GA complex directly interacts with SLENDER RICE1 (SLR1), a DELLA repressor protein in GA signaling. Here, we demonstrated, by pull-down and bimolecular fluorescence complementation (BiFC) experiments, that the GA-dependent GID1-SLR1 interaction also occurs in planta. GA(4) was found to have the highest affinity to GID1 in Y2H assays and is the most effective form of GA in planta. Domain analyses of SLR1 using Y2H, gel filtration, and BiFC methods revealed that the DELLA and TVHYNP domains of SLR1 are required for the GID1-SLR1 interaction. To identify the important regions of GID1 for GA and SLR1 interactions, we used many different mutant versions of GID1, such as the spontaneous mutant GID1s, N- and C-terminal truncated GID1s, and mutagenized GID1 proteins with conserved amino acids replaced with Ala. The amino acid residues important for SLR1 interaction completely overlapped the residues required for GA binding that were scattered throughout the GID1 molecule. When we plotted these residues on the GID1 structure predicted by analogy with HSL tertiary structure, many residues were located at regions corresponding to the substrate binding pocket and lid. Furthermore, the GA-GID1 interaction was stabilized by SLR1. Based on these observations, we proposed a molecular model for interaction between GA, GID1, and SLR1.

Multiple loss-of-function of Arabidopsis gibberellin receptor AtGID1s completely shuts down a gibberellin signal.

Arabidopsis carries three receptor genes for the phytohormone gibberellin (GA), AtGID1a, AtGID1b and AtGID1c. Expression of each gene in the rice gid1-1 mutant for GA receptors causes reversion of its severely dwarfed phenotype and GA insensitivity to a normal level, even though each loss-of-function mutant shows no clear phenotype in Arabidopsis (Nakajima et al., 2006). In this paper, we report the functional redundancy and specificity of each AtGID1 by analyzing the multiple mutants for loss of function. Seeds of the double knockout mutants atgid1a atgid1b, atgid1a atgid1c and atgid1b atgid1c germinated normally. The double knockout mutant atgid1a atgid1c showed a dwarf phenotype, while other double mutants were of normal height compared to the wild-type. The stamens of the double knockout mutant atgid1a atgid1b were significantly shorter than those of the wild-type, and this leads to low fertility. A severe disarrangement of the pattern on its seed surface was also observed. The triple knockout mutant atgid1a atgid1b atgid1c did not germinate voluntarily, and only started to grow when the seed coat was peeled off after soaking. Seedlings of the triple knockout mutants were severe dwarfs, only a few millimeters high after growing for 1 month. Moreover, the triple knockout seedlings completely lost their ability to respond to exogenously applied GA. These results show that all AtGID1s function as GA receptors in Arabidopsis, but have specific role(s) for growth and development.

Highly sensitive quantitative analysis of nicotianamine using LC/ESI-TOF-MS with an internal standard.

A highly sensitive quantitative method for analyzing nicotianamine (NA) by liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) is reported. Fluorenylmethoxycarbonylation of nicotianamine reduced its polarity and enabled its retention in a reversed-phase column. The adoption of N(epsilon)-nicotyllysine (NL) as an internal standard ensured reliable quantification by giving a linear calibration curve drawn between the NA/NL molar ratios of standard solutions injected and the NA/NL area ratios in mass chromatograms. The high sensitivity of this analytical method allowed us to measure the amount of NA. This analytical method has applications to all research concerning NA.

Sequential regulation of gibberellin, brassinosteroid, and jasmonic acid biosynthesis occurs in rice coleoptiles to control the transcript levels of anti-microbial thionin genes.

Transcripts of thionin genes encoding antimicrobial peptides were present at a high level in rice coleoptiles just after germination, and decreased to an undetectable level after about 3 d, but this decline was suppressed by co-treatment with gibberellic acid (GA(3)) and brassinolide (BL). The temporal expression patterns of key enzyme genes for the biosyntheses of gibberellins (GAs) and brassinosteroids (BRs) were correlated with the fluctuation of thionin mRNAs. Jasmonic acid (JA) replaced the effect of GA3 and BL, and its change in endogenous level was parallel to that of the thionin genes. These results strongly suggest that thionin gene expression was positively regulated by JA, whose endogenous level was synergistically regulated by GAs and BRs. In contrast, thionin gene expression in etiolated seedlings remained high while the endogenous level of JA was low, suggesting the presence of another signaling pathway in the dark to maintain the thionin level.

Identification of a peptide mimic of bioactive gibberellins with affinity to GA 2-oxidase.

Previously we reported the first example of peptide mimics of a small hydrophobic molecule, a phytohormone gibberellin. The second peptide mimic of gibberellin has been identified from random peptide libraries by its affinity to a type of catalyzing enzyme of gibberellins, which specifically recognizes bioactive gibberellins. These results suggest that even hydrophobic compounds can be mimicked by peptides.

Identification and characterization of Arabidopsis gibberellin receptors.

Three gibberellin (GA) receptor genes (AtGID1a, AtGID1b and AtGID1c), each an ortholog of the rice GA receptor gene (OsGID1), were cloned from Arabidopsis, and the characteristics of their recombinant proteins were examined. The GA-binding activities of the three recombinant proteins were confirmed by an in vitro assay. Biochemical analyses revealed similar ligand selectivity among the recombinants, and all recombinants showed higher affinity to GA(4) than to other GAs. AtGID1b was unique in its binding affinity to GA(4) and in its pH dependence when compared with the other two, by only showing binding in a narrow pH range (pH 6.4-7.5) with 10-fold higher affinity (apparent K(d) for GA(4) = 3 x 10(-8) m) than AtGID1a and AtGID1c. A two-hybrid yeast system only showed in vivo interaction in the presence of GA(4) between each AtGID1 and the Arabidopsis DELLA proteins (AtDELLAs), negative regulators of GA signaling. For this interaction with AtDELLAs, AtGID1b required only one-tenth of the amount of GA(4) that was necessary for interaction between the other AtGID1s and AtDELLAs, reflecting its lower K(d) value. AtDELLA boosted the GA-binding activity of AtGID1 in vitro, which suggests the formation of a complex between AtDELLA and AtGID1-GA that binds AtGID1 to GA more tightly. The expression of each AtGID1 clone in the rice gid1-1 mutant rescued the GA-insensitive dwarf phenotype. These results demonstrate that all three AtGID1s functioned as GA receptors in Arabidopsis.

AtFLA11, a fasciclin-like arabinogalactan-protein, specifically localized in sclerenchyma cells.

An arabinogalactan-protein macroarray of all 48 Arabidopsis arabinogalactan-protein genes was prepared as a handy detection system for arabinogalactan-protein gene expression. The major transcript in inflorescence stems was identified as AtFLA11. AtFLA11 is categorized as a fasciclin-like arabinogalactan-protein that possesses a fasciclin domain with a cell adhesion function in animal cells. AtFLA11 was specifically expressed in the sclerenchyma cells of inflorescence stems and siliques, which are characterized by their thick secondary cell walls, and was confirmed by immunostaining at the protein level. The fluctuation of AtFLA11 transcripts during the maturation process of sclerenchyma cells suggests its role in the formation of the secondary cell wall.

Structure, epitope mapping, and docking simulation of a gibberellin mimic peptide as a peptidyl mimotope for a hydrophobic ligand.

Using NMR spectroscopy and simulated annealing calculations, we determined the solution structure of the disulfide-linked cyclized decapeptide ACLPWSDGPC (SD), which is bound to an anti-(gibberellin A(4)) mAb 4-B8(8)/E9 and was found to be the first peptidyl mimotope for a hydrophobic ligand. The resulting structure of the peptide showed a beta-turn-like conformation in residues three to seven and the region converges well (average rmsd 0.54 A). The binding activity and the epitopes of the peptide to the antibody were assessed using saturation transfer difference (STD)-NMR experiments. We also conducted docking simulations between the peptide and the mAb to determine how the peptide is bound to the mAb. Resonances around the beta-turn-like conformation of peptide SD (residues 3-5) showed strong STD enhancement, which agreed well with results from docking simulation between peptide SD and the mAb. Together with the commonality of amino acid residues of the mAb involved in interactions with gibberellin A(4) (GA(4)) and peptide SD, we concluded that peptide SD is bound to the antigen-binding site of mAb 4-B8(8)/E9 as a GA(4) mimic, confirming evidence for the existence of peptide mimics even for hydrophobic ligands.

GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin.

Gibberellins (GAs) are phytohormones that are essential for many developmental processes in plants. It has been postulated that plants have both membrane-bound and soluble GA receptors; however, no GA receptors have yet been identified. Here we report the isolation and characterization of a new GA-insensitive dwarf mutant of rice, gid1. The GID1 gene encodes an unknown protein with similarity to the hormone-sensitive lipases, and we observed preferential localization of a GID1-green fluorescent protein (GFP) signal in nuclei. Recombinant glutathione S-transferase (GST)-GID1 had a high affinity only for biologically active GAs, whereas mutated GST-GID1 corresponding to three gid1 alleles had no GA-binding affinity. The dissociation constant for GA4 was estimated to be around 10(-7) M, enough to account for the GA dependency of shoot elongation. Moreover, GID1 bound to SLR1, a rice DELLA protein, in a GA-dependent manner in yeast cells. GID1 overexpression resulted in a GA-hypersensitive phenotype. Together, our results indicate that GID1 is a soluble receptor mediating GA signalling in rice.

Gibberellin 2-oxidases from seedlings of adzuki bean (Vigna angularis) show high gibberellin-binding activity in the presence of 2-oxoglutarate and Co2+.

Five full-length cDNA encoding gibberellin 2-oxidases, VaGA2oxA1, VaGA2oxA2, VaGA2oxB1, VaGA2oxB2, and VaGA2oxB3, were cloned from etiolated adzuki bean (Vigna angularis cv. Dainagon) seedlings, and their enzymatic characteristics were examined using recombinant enzymes fused with glutathione S-transferase (GST). Recombinant VaGA2oxA1 (rVaGA2oxA1) and rVaGA2oxA2 showed 2beta-hydroxylation activity by converting GA1, GA4, GA9, GA20, GA4-methyl ester, and 16,17-dihydro-GA4 to the corresponding 2beta-hydroxylated gibberellins, which were identified by GC/MS. rVaGA2oxB1, rVaGA2oxB2, and rVaGA2oxB3 showed similar activity by converting [3H4]-16,17-dihydro-GA4 to a metabolite showing an Rf value of 16,17-dihydro-GA34. RNA-blot analysis showed that VaGA2oxA1 and VaGA2oxA2 were the major ones expressed in etiolated hypocotyls. The addition of Co2+ instead of Fe2+ to the assay medium apparently reduced the enzymatic activity, but increased the binding of [3H4]-16,17-dihydro-GA4 to rVaGA2oxA1, indicating the possibility that VaGA2oxs can be detected as gibberellin-binding proteins under certain conditions.

Similarities and differences between the characteristics of gibberellin-binding protein and gibberellin 2-oxidases in adzuki bean (Vigna angularis) seedlings.

Gibberellin-binding proteins (GBPs) were purified ca. 230,000 fold. The characteristics of adzuki GBP were examined and compared with those of a recombinant gibberellin 2-oxidase (rVaGA2oxA1) that was fused with glutathione S-transferase (GST). VaGA2oxA1 was most abundantly expressed in etiolated adzuki bean seedlings, and VaGA2oxA1 and GBPs from adzuki bean seedlings showed gibberellin-binding activity when incubated with 2-oxoglutarate and Co2+. Both rVaGA2oxA1 and partially purified GBPs from adzuki bean seedlings showed very similar selectivity to gibberellins in binding assays, where biologically active gibberellins such as GA4, GA3, GA7, and GA1 showed higher binding affinity than biologically inactive gibberellins such as GA8, GA34, and 3-epi-GA4. The polyclonal antibody raised against rVaGA2oxA1 cross-reacted with all rVaGA2oxs (rVaGA2oxA1, rVaGA2oxA2, rVaGA2oxB1, rVaGA2oxB2, and rVaGA2oxB3) whose cDNAs were cloned from adzuki bean seedlings. Treated with the antibody, the recombinants that originally showed gibberellin-binding activity lost both binding activity and enzymatic activity. In contrast to the recombinants, the gibberellin-binding activity of GBPs from adzuki bean seedlings was hardly affected by the antibody treatment. The GBPs showed very weak gibberellin 2-oxidase-like activity, and it was not affected by the antibody treatment either. These observations suggest that a major component that showed GA-binding activity was apparently different from any gibberellin 2-oxidase cloned from the seedlings.