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.     

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.     

Proteolysis-independent downregulation of DELLA repression in Arabidopsis by the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1

This article presents evidence that DELLA repression of gibberellin (GA) signaling is relieved both by proteolysis-dependent and -independent pathways in Arabidopsis thaliana. DELLA proteins are negative regulators of GA responses, including seed germination, stem elongation, and fertility. GA stimulates GA responses by causing DELLA repressor degradation via the ubiquitin-proteasome pathway. DELLA degradation requires GA biosynthesis, three functionally redundant GA receptors GIBBERELLIN INSENSITIVE DWARF1 (GID1a, b, and c), and the SLEEPY1 (SLY1) F-box subunit of an SCF E3 ubiquitin ligase. The sly1 mutants accumulate more DELLA proteins but display less severe dwarf and germination phenotypes than the GA biosynthesis mutant ga1-3 or the gid1abc triple mutant. Interestingly, GID1 overexpression rescued the sly1 dwarf and infertility phenotypes without decreasing the accumulation of the DELLA protein REPRESSOR OF ga1-3. GID1 rescue of sly1 mutants was dependent on the level of GID1 protein, GA, and the presence of a functional DELLA motif. Since DELLA shows increasing interaction with GID1 with increasing GA levels, it appears that GA-bound GID1 can block DELLA repressor activity by direct protein-protein interaction with the DELLA domain. Thus, a SLY1-independent mechanism for GA signaling may function without DELLA degradation.     

Functional analysis of beta- and epsilon-ring carotenoid hydroxylases in Arabidopsis

Lutein and zeaxanthin are dihydroxy xanthophylls that are produced from their corresponding carotene precursors by the action of beta- and epsilon -ring carotenoid hydroxylases. Two genes that encode beta-ring hydroxylases (beta-hydroxylases 1 and 2) have been identified in the Arabidopsis genome and are highly active toward beta-rings but only weakly active toward epsilon -rings. A third distinct activity required for epsilon -ring hydroxylation has been defined by mutation of the LUTEIN1 (LUT1) locus, but LUT1 has not yet been cloned. To address the individual and overlapping functions of the three Arabidopsis carotenoid hydroxylase activities in vivo, T-DNA knockout mutants corresponding to beta-hydroxylases 1 and 2 (b1 and b2, respectively) were isolated and all possible hydroxylase mutant combinations were generated. beta-Hydroxylase single mutants do not exhibit obvious growth defects and have limited impact on carotenoid composition relative to the wild type, suggesting that the encoded proteins have a significant degree of functional redundancy in vivo. Surprisingly, the b1 b2 double mutant, which lacks both known beta-hydroxylase enzymes, still contains significant levels of beta-carotene-derived xanthophylls, suggesting that additional beta-ring hydroxylation activity exists in vivo. The phenotype of double and triple hydroxylase mutants indicates that at least a portion of this activity resides in the LUT1 gene product. Despite the severe reduction of beta-carotene-derived xanthophylls (up to 90% in the lut1 b1 b2 triple mutant), the double and triple hydroxylase mutants still contain at least 50% of the wild-type amount of hydroxylated beta-rings. This finding suggests that it is the presence of minimal amounts of hydroxylated beta-rings, rather than minimal amounts of specific beta-carotene-derived xanthophylls, that are essential for light-harvesting complex II assembly and function in vivo. The carotenoid profiles in wild-type seeds and the effect of single and multiple hydroxylase mutations are distinct from those in photosynthetic tissues, indicating that the activities of each gene product differ in the two tissues. Overall, the hydroxylase mutants provide insight into the unexpected overlapping activity of carotenoid hydroxylases in vivo.     

The New Rga Locus Encodes a Negative Regulator of Gibberellin Response in Arabidopsis Thaliana

We have identified a new locus involved in gibberellin (GA) signal transduction by screening for suppressors of the Arabidopsis thaliana GA biosynthetic mutant ga1-3. The locus is named RGA for repressor of ga1-3. Based on the recessive phenotype of the digenic rga/ga1-3 mutant, the wild-type gene product of RGA is probably a negative regulator of GA responses. Our screen for suppressors of ga1-3 identified 17 mutant alleles of RGA as well as 10 new mutant alleles at the previously identified SPY locus. The digenic (double homozygous) rga/ga1-3 mutants are able to partially repress several defects of ga1-3 including stem growth, leaf abaxial trichome initiation, flowering time, and apical dominance. The phenotype of the trigenic mutant (triple homozygous) rga/spy/ga1-3 shows that rga and spy have additive effects regulating flowering time, abaxial leaf trichome initiation and apical dominance. This trigenic mutant is similar to wild type with respect to each of these developmental events. Because rga/spy/ga1-3 is almost insensitive to GA for hypocotyl growth and its bolting stem is taller than the wild-type plant, the combined effects of the rga and spy mutations appear to allow GA-independent stem growth. Our studies indicate that RGA lies on a separate branch of the GA signal transduction pathway from SPY, which leads us to propose a modified model of the GA response pathway.     

Arabidopsis extra-large G proteins (XLGs) regulate root morphogenesis

As in mammalian systems, heterotrimeric G proteins, composed of alpha, beta and gamma subunits, are present in plants and are involved in the regulation of development and cell signaling. Besides the sole prototypical G protein alpha subunit gene, GPA1, the Arabidopsis thaliana genome has three extra-large GTP-binding protein (XLG)-encoding genes: XLG1 (At2g23460), XLG2 (At4g34390) and XLG3 (At1g31930). The C-termini of the XLGs are Galpha domains that are homologous to GPA1, whereas their N-termini each contain a cysteine-rich region and a putative nuclear localization signal (NLS). GFP fusions with each XLG confirmed nuclear localization. All three XLG genes are expressed in essentially all plant organs, with strong expression in vascular tissues, primary root meristems and lateral root primordia. Analysis of single, double and triple T-DNA insertional mutants of the XLG genes revealed redundancy in XLG function. Dark-grown xlg1-1 xlg2-1 xlg3-1 triple mutant plants showed markedly increased primary root length compared with wild-type plants. This phenotype was not observed in dark-grown xlg single mutants, and was suppressed upon complementation of the xlg triple mutant with each XLG. Root cell sizes of the xlg triple mutant and root morphology were highly similar to those of wild-type roots, suggesting that XLGs may regulate cell proliferation. Dark-grown roots of the xlg triple mutants also showed altered sensitivity to sugars, ABA hyposensitivity and ethylene hypersensitivity, whereas seed germination in xlg triple mutants was hypersensitive to osmotic stress and ABA. As plant-specific proteins, regulatory mechanisms of XLGs may differ from those of conventional Galphas.     

Gibberellin requirement for Arabidopsis seed germination is determined both by testa characteristics and embryonic abscisic acid

The mechanisms imposing a gibberellin (GA) requirement to promote the germination of dormant and non-dormant Arabidopsis seeds were analyzed using the GA-deficient mutant ga1, several seed coat pigmentation and structure mutants, and the abscisic acid (ABA)-deficient mutant aba1. Testa mutants, which exhibit reduced seed dormancy, were not resistant to GA biosynthesis inhibitors such as tetcyclacis and paclobutrazol, contrarily to what was found before for other non-dormant mutants in Arabidopsis. However, testa mutants were more sensitive to exogenous GAs than the wild-types in the presence of the inhibitors or when transferred to a GA-deficient background. The germination capacity of the ga1-1 mutant could be integrally restored, without the help of exogenous GAs, by removing the envelopes or by transferring the mutation to a tt background (tt4 and ttg1). The double mutants still required light and chilling for dormancy breaking, which may indicate that both agents can have an effect independently of GA biosynthesis. The ABA biosynthesis inhibitor norflurazon was partially efficient in releasing the dormancy of wild-type and mutant seeds. These results suggest that GAs are required to overcome the germination constraints imposed both by the seed coat and ABA-related embryo dormancy.     

ATMYB123和ATKOR1基因参与拟南芥根发育的调控

从拟南芥T-DNA插入突变体库中筛选到2个根发育相关基因ATMYB123和ATKOR1表达缺失的突变体atmyb123和atkor1,通过杂交构建这两个基因表达缺失的双突变体atmyb123/atkor1,以明确这两个基因在根发育中的作用。结果显示:(1)ATMYB123表达缺失突变体atmyb123植株地上部分发育减缓,种皮颜色变黄,而ATKOR1表达缺失突变体atkor1植株在这两方面与其野生型没有明显差异;两基因缺失均显著影响了拟南芥根的发育,根生长受到了严重抑制。(2)双突变体atmyb123/atkor1在植株形态和种皮颜色方面表现出单突变体AT-MYB123的特点,而其根长却介于两单突变体的中间。(3)进一步研究发现,培养基pH改变、NaCl处理、外源GA施用均没有改变突变体根生长趋势,显示这3种因素与两基因缺失突变引起的根发育抑制无关。研究表明,AT-MYB123和ATKOR1基因参与拟南芥根的发育调控,转录因子ATMYB123可能作为主调控因子参与ATKOR1对拟南芥根发育的调控。     

The DELLA domain of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A gibberellin receptor of Arabidopsis

Gibberellic acid (GA) promotes seed germination, elongation growth, and flowering time in plants. GA responses are repressed by DELLA proteins, which contain an N-terminal DELLA domain essential for GA-dependent proteasomal degradation of DELLA repressors. Mutations of or within the DELLA domain of DELLA repressors have been described for species including Arabidopsis thaliana, wheat (Triticum aestivum), maize (Zea mays), and barley (Hordeum vulgare), and we show that these mutations confer GA insensitivity when introduced into the Arabidopsis GA INSENSITIVE (GAI) DELLA repressor. We also demonstrate that Arabidopsis mutants lacking the three GA INSENSITIVE DWARF1 (GID1) GA receptor genes are GA insensitive with respect to GA-promoted growth responses, GA-promoted DELLA repressor degradation, and GA-regulated gene expression. Our genetic interaction studies indicate that GAI and its close homolog REPRESSOR OF ga1-3 are the major growth repressors in a GA receptor mutant background. We further demonstrate that the GA insensitivity of the GAI DELLA domain mutants is explained in all cases by the inability of the mutant proteins to interact with the GID1A GA receptor. Since we found that the GAI DELLA domain alone can mediate GA-dependent GID1A interactions, we propose that the DELLA domain functions as a receiver domain for activated GA receptors.     

Characterization of a helminthosporic acid analog that is a selective agonist of gibberellin receptor

Helminthosporol, a natural growth regulator isolated from a fungus, stimulates hypocotyl growth and seed germination, similar to gibberellin (GA). We recently reported that helminthosporic acid (H-acid), a synthetic analog of helminthosporol, acts as an agonist of GA receptor. In this study, we showed that a H-acid analog, in which the hydroxymethyl group at the C-8 position of H-acid was converted to a keto group, acts as a selective GA receptor agonist. 1) This analog shows higher hypocotyl elongation activity in Arabidopsis than H-acid does, and induces the degradation of DELLA protein and 2) leads to the formation of the GID1-DELLA complex and 3) regulates the expression of GA-related genes. In addition, 4) its hypocotyl elongation activity was not observed in a atgid1a single mutant, and 5) this analog could promote only the interaction between specific GA receptors and DELLA proteins in vitro. Taken together, our results strongly suggest that the selectivity of the reported H-acid analog depends on the specificity of its GA receptor binding activity.     

Effects of the gibberellin biosynthetic inhibitor uniconazol on mutants of Arabidopsis

Using the gibberellin (GA) biosynthetic inhibitor Uniconazol, we determined that det1, a mutant that no longer requires light to be germinated, still requires GA synthesis for germination. This result suggests that dark inhibition of germination in Arabidopsis may be due to inhibition of GA synthesis by the DET1 gene product in mature wild-type seeds. Similar experiments with mutants that lack seed dormancy due to a reduced sensitivity to abscisic acid (abi) have shown that abi1 and abi3 no longer require GA for germination. Furthermore, by shifting wild-type seeds to inhibitor at 6-hour intervals during imbibition, we determined that GA synthesis is only required during the first 24 hours of the imbibition process to reverse abscisic acid-induced dormancy in Arabidopsis.     

Characterization of mutants with reduced seed dormancy at two novel rdo loci and a further characterization of rdo1 and rdo2 in Arabidopsis

Seed dormancy and germination are complex traits that are controlled by many genes. Four mutants in Arabidopsis thaliana exhibiting a reduced dormancy phenotype, designated rdo1, rdo2, rdo3 , and rdo4, have been characterized, both genetically and physiologically. Two of these mutants, rdo1 and rdo2 , have been described before, the other two represent novel loci. The mutants mapped on chromosome 1 ( rdo3 ), chromosome 2 ( rdo2 and rdo4 ), and chromosome 3 ( rdo1 ). None of these loci has been related to dormancy before. All four mutants show pleiotropic effects in the adult plant stage, which are different for each mutant. None of the mutants is deficient in ABA. Compared to L er (wild-type), ABA sensitivity is not altered either, thereby excluding the possibility that ABA is involved in causing the reduced dormancy phenotype. The GA requirement was studied by using the GA biosynthesis inhibitor paclobutrazol, and genetically by generating double mutants with the GA-deficient mutant ga1-3 . The results obtained by these two methods were comparable for all but one mutant: rdo1 . In a GA-deficient background, rdo1 , rdo2 and rdo3 , all show sensitivity to GA between that of ga1-3 and ga1-3 aba1. However, when using paclobutrazol rdo1 exhibited the same sensitivity as rdo4 and wild-type. Analysis of double mutants among the rdo mutants revealed a very complex and inconsistent pattern.     

Casein kinase II α subunits affect multiple developmental and stress-responsive pathways in Arabidopsis

Casein kinase II (formerly known as CK2), a ubiquitous Ser/Thr kinase, plays critical roles in all higher organisms including plants. The CK2 holoenzyme consists of two catalytic α subunits and two regulatory β subunits. The Arabidopsis genome has four α subunit and four β subunit genes, and members of both the α and β subunit families have been shown to be localized in the cytoplasm, nucleus and also in chloroplasts. However, the biological roles of CK2 subunits have not been fully characterized yet. Here we identified T-DNA insertion mutants in three α subunit genes (α1, α2 and α3) and made double and triple mutants. The CK2 α1α2α3 triple mutants displayed reduced CK2 activity compared with wild-type seedlings. Phenotypic characterization showed that CK2 α1α2α3 triple mutants are late flowering under both long- and short-day conditions. Genes encoding floral integrators are differentially regulated in the triple mutant compared with the wild-type plants. CK2 α1α2α3 triple mutants also displayed reduced hypocotyl growth, smaller cotyledon size and a reduced number of lateral roots compared with wild-type seedlings under light. Abscisic acid-induced blockage of seed germination and cotyledon greening is reduced in CK2 α subunit mutants in an additive manner. Moreover, CK2 α subunit mutants are also hyposensitive to a NaCl-induced blockage of seed germination. Taken together, these data suggest that CK2 α subunits affect diverse developmental and stress responsive pathways in Arabidopsis.     

Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants

Degradation of active C(19)-gibberellins (GAs) by dioxygenases through 2beta-hydroxylation yields inactive GA products. We identified two genes in Arabidopsis (AtGA2ox7 and AtGA2ox8), using an activation-tagging mutant screen, that encode 2beta-hydroxylases. GA levels in both activation-tagged lines were reduced significantly, and the lines displayed dwarf phenotypes typical of mutants with a GA deficiency. Increased expression of either AtGA2ox7 or AtGA2ox8 also caused a dwarf phenotype in tobacco, indicating that the substrates for these enzymes are conserved. AtGA2ox7 and AtGA2ox8 are more similar to each other than to other proteins encoded in the Arabidopsis genome, indicating that they may constitute a separate class of GA-modifying enzymes. Indeed, enzymatic assays demonstrated that AtGA2ox7 and AtGA2ox8 both perform the same GA modification: 2beta-hydroxylation of C(20)-GAs but not of C(19)-GAs. Lines containing increased expression of AtGA2ox8 exhibited a GA dose-response curve for stem elongation similar to that of the biosynthetic mutant ga1-11. Double loss-of-function Atga2ox7 Atga2ox8 mutants had twofold to fourfold higher levels of active GAs and displayed phenotypes associated with excess GAs, such as early bolting in short days, resistance to the GA biosynthesis inhibitor ancymidol, and decreased mRNA levels of AtGA20ox1, a gene in the GA biosynthetic pathway.     

Ethylene stimulates nutations that are dependent on the ETR1 receptor

Ethylene influences a number of processes in Arabidopsis (Arabidopsis thaliana) through the action of five receptors. In this study, we used high-resolution, time-lapse imaging to examine the long-term effects of ethylene on growing, etiolated Arabidopsis seedlings. These measurements revealed that ethylene stimulates nutations of the hypocotyls with an average delay in onset of over 6 h. The nutation response was constitutive in ctr1-2 mutants maintained in air, whereas ein2-1 mutants failed to nutate when treated with ethylene. Ethylene-stimulated nutations were also eliminated in etr1-7 loss-of-function mutants. Transformation of the etr1-7 mutant with a wild-type genomic ETR1 transgene rescued the nutation phenotype, further supporting a requirement for ETR1. Loss-of-function mutations in the other receptor isoforms had no effect on ethylene-stimulated nutations. However, the double ers1-2 ers2-3 and triple etr2-3 ers2-3 ein4-4 loss-of-function mutants constitutively nutated in air. These results support a model where all the receptors are involved in ethylene-stimulated nutations, but the ETR1 receptor is required and has a contrasting role from the other receptor isoforms in this nutation phenotype. Naphthylphthalamic acid eliminated ethylene-stimulated nutations but had no effect on growth inhibition caused by ethylene, pointing to a role for auxin transport in the nutation phenotype.     

The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis.

The first committed step in the gibberellin (GA) biosynthetic pathway is the conversion of geranylgeranyl pyrophosphate (GGPP) through copalyl pyrophosphate (CPP) to ent-kaurene catalyzed by ent-kaurene synthetases A and B. The ga1 mutants of Arabidopsis are gibberellin-responsive male-sterile dwarfs. Biochemical studies indicate that biosynthesis of GAs in the ga1 mutants is blocked prior to the synthesis of ent-kaurene. The GA1 locus was cloned previously using the technique of genomic subtraction. Here, we report the isolation of a nearly full-length GA1 cDNA clone from wild-type Arabidopsis. This cDNA clone encodes an active protein and is able to complement the dwarf phenotype in ga1-3 mutants by Agrobacterium-mediated transformation. In Escherichia coli cells that express both the Arabidopsis GA1 gene and the Erwinia uredovora gene encoding GGPP synthase, CPP was accumulated. This result indicates that the GA1 gene encodes the enzyme ent-kaurene synthetase A, which catalyzes the conversion of GGPP to CPP. Subcellular localization of the GA1 protein was studied using 35S-labeled GA1 protein and isolated pea chloroplasts. The results showed that the GA1 protein is imported into and processed in pea chloroplasts in vitro.