1. History
Gibberellins are plant hormones, tetracyclic diterpene carboxylic acids that were discovered in 1926 as a phytotoxin produced by the fungus Gibberella fujikuroi. The latter caused a pathological longitudinal growth in rice called "foolish seedling disease" (1). The active compound was isolated from the fungus in 1930 by Yabuta and Sumiki (2) and was called gibberellin. In 1958, a first gibberellin (GA 1) was purified from runner bean seeds (Phaseolus coccineus) by MacMillan and coworkers (3). Since this discovery, 112 gibberellins have been identified (4).
2. Biosynthesis and Metabolism
Gibberellins are isoprenoid compounds synthesized from C2 units by the mevalonic acid pathway. Geranyl geranyl diphosphate, a C20 molecule, serves as a donor for the entire carbon skeleton of gibberellins. Starting from geranyl geranyl diphosphate, biosynthesis of gibberellins can be divided into three stages according to the nature of the enzymes involved and their subcellular localization (Fig. 1). The pathway is well-characterized, owing to the existence of gibberellin-deficient mutants that are easily identified by their dwarfed stature (5, 6). Gibberellin-deficient mutants are blocked at a particular step in the pathway, and their phenotype can be reverted by external addition of active gibberellins.
Figure 1. Gibberellin biosynthetic pathway in higher plants. Steps involving GA1, GA2, GA4, and GA5 in Arabidopis are indicated.
Several genes encoding gibberellin-biosynthetic enzymes have been characterized, and their products seem to catalyze multiple steps. First, geranyl geranyl diphosphate is converted into ent-kaurene by two cyclization steps. The enzymes catalyzing these reactions are ent-kaurene synthase A and B, also termed ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) (7). The gene encoding the CPS has been cloned from Arabidopsis (GA1) (8), maize (Anther ear-1, An1) (9), pea (LS) (10), tomato (11), and pumpkin (12), whereas that for KS was cloned from pumpkin endosperm (13) and from Arabidopsis ( GA2) (14).
The KS activity is mostly associated with stroma of proplastids in rapidly dividing plant tissues (15); however, GA1 promoter activity is also present in fully expanded leaves (16), suggesting that gibberellins are transported to responsive tissues. In Arabidopsis Arabidopsis, GA1 gene expression appears to be highly regulated during growth and development. In addition, GA1 promoter activity was restricted to specific cell types, such as shoot apices, root tips, developing seeds and flowers, and vascular tissue of mature leaves (17).
The second stage converts ent-kaurene to GA-aldehyde. Ent-kaurene is subjected to five sequential oxidation steps, resulting in GA^-aldehyde. These reactions take place in the endoplasmic reticulum and seem to be catalyzed by a single membrane-associated cytochrome P450 monooxygenase. The gene encoding this enzyme was recently cloned from maize (Dwarf-3, D3) (18). Its expression was observed in seedlings, developing leaves, vegetative meristems, and roots. The predicted protein carries the Fe-binding domain that is characteristic of cytochrome P450 proteins (18).
The third stage converts GA^-aldehyde to various gibberellins and takes place in the cytoplasm.
The first step in this process consists of the oxidation of GA^-aldehyde to GA^. Subsequently,GA^ is subjected to various reactions, depending on the organism. In the past, genes encoding one of the major enzymes involved in these reactions, GA 20-oxidase, were characterized in different species such as pumpkin (19), Arabidopsis (GA5) (20), and spinach (21). GA 20-oxidases belong to the family of 2-oxoglutarate-dependent dioxygenases. In Arabidopsis, GA 20-oxidases are encoded by a small multigene family. Expression of the GA5 gene in leaves was enhanced when plants were shifted from short-day to long-day conditions, indicating photoperiod regulation. In addition, GA5 was down-regulated by GA4 treatment, suggesting feedback repression of the pathway (20). Finally,a 3b-hydroxylase catalyzes the formation of biologically active gibberellins. The GA4 locus encoding a 3b-hydroxylase was recently cloned from Arabidopsis (22). The gene is ubiquitously expressed, but its messenger RNA is most abundant in siliques. As with GA5, GA4 is also subjected to a feedback regulatory mechanism (23).
Relatively few gibberellins appear to be biologically active, such as GA!, GA3, and GA4. The remaining ones are the precursors or the deactivated forms of active gibberellins. The latter are generally deactivated by 2b-hydroxylation. The only deactivation mutant described thus far is the slender (sln) genotype of Pisum sativum . A mutation at this locus is responsible for a large accumulation of GA20, the precursor of GA1, and gives rise to the elongated phenotype. It was suggested that sln encodes a regulatory protein controlling two genes involved in the degradation pathway (24). Another process in gibberellin metabolism is their conjugation to inactive derivatives, including glucosyl ethers and glucose esters, that are found mainly in seeds (25).
3. Signal Perception and Transduction
The gibberellin biosynthesis pathway is well-characterized, but much less is known about how gibberellins are perceived by the plant and how the signal is transduced to control the expression of gibberellin-regulated genes (26, 27). Biochemical studies on barley aleurone protoplasts suggested that gibberellin binds to a receptor located at the external face of the plasma membrane (28). This receptor has not yet been isolated, however, nor has any gibberellin receptor been cloned to date.
Our current knowledge of gibberellin signaling is based mainly on the characterization of four mutants of Arabidopsis: spy ( spindly), gai (gibberellin-insensitive ), rga (repressor of ga1-3), and pkl (pickle ). None of these mutants can be entirely reverted by exogenous application of gibberellin, supporting their role in gibberellin signaling.
The spy mutants were screened for their ability to germinate in the presence of the gibberellin biosynthesis inhibitor paclobutrazol, which blocks the cytochrome P450 monooxygenase involved in the oxidation of ent-kaurene to ent-kaurenoic acid (29). Spy mutants phenocopy wild-type plants that were repeatedly treated with GA3, presenting a constitutive gibberellin-response phenotype that is characterized by long hypocotyls, pale green foliage, increased stem elongation, early flowering, partial male sterility, and parthenocarpic fruit development. Moreover, spy plants remain gibberellin-responsive. Spy is partially epistatic to ga1-2. Collectively, these data indicate that SPY is a negative regulator of at least one branch in gibberellin signaling.
The SPY gene was recently cloned using a T-DNA tagging approach (30). The predicted SPY protein contains a tetratricopeptide repeat of 34 amino acid residues in its N-terminal region. This motif is also found in some other eukaryotic and prokaryotic proteins and is proposed to form an amphipathic a-helix that mediates protein-protein interactions (31). The tetratricopeptide repeat appears to be important for the normal function of SPY, because some of the mutant phenotypes result from a deletion in this motif. The C-terminal region of SPY shows sequence similarity to the mammalian Ser/Thr O-linked #-acetylglucosamine (O-GlcNAc) transferase, which plays an important role in the regulation of the activity of various nuclear and cytosolic proteins, either directly or by inhibiting their phosphorylation (32, 33). Because certain spy alleles are affected in the region bearing similarity to O-GlcNAc transferase, this activity seems to be involved in proper functioning of the SPY gene product.
A second player is GAI. Gai mutant plants present a dwarf phenotype (reduced plant height, decrease in apical dominance, and limited seed germination), comparable to gibberellin-deficient plants. The lack of response of gai mutants to exogenous application of gibberellin, however, suggests that the GAI protein acts in signal transduction (34, 35). Gai heterozygotes show an intermediate mutant phenotype, indicating that the mutation is semidominant.
The GAI gene was recently isolated using Ds transposon tagging (36). In addition, a closely related gene named GRS (GAI-relatedsequence) was cloned (36). Comparison of the predicted amino acid sequences of the GRS and GAI proteins with the sequence databases showed that they are members of the VHIID family of plant transcription factors defined by Di Laurenzio et al. (37). A first member of this novel class of transcription factors was called SCARECROW (SCR) and controls cell fate in Arabidopsis roots. The VHIID proteins are putative transcription factors that contain three main motifs: a central -Val-His-Ile-Ile-Asp-sequence, with a Leu-X-X-Leu-Leu-sequence (where X represents any amino acid residue) in its immediate vicinity, and an -Arg-Val-Glu-Arg-sequence in the C-terminal region (38). The Leu-X-X-Leu-Leu-motif is a signature motif in transcriptional coactivators that mediates binding to nuclear receptors (39). The GAI protein possesses a fourth sequence in its N-terminal region (-Asp-Glu-Leu-Leu-Ala-) that appears important for its normal function. A nearby Ser/Thr-rich stretch is a potential target of SPY action. GAI was proposed to act directly as a transcriptional repressor of target genes that promote gibberellin-mediated processes, or indirectly as a transcriptional activator that induces the expression of such a repressor (36). This hypothesis implies that gibberellin would modulate the pathway by derepression and, therefore, by inactivation of GAI. An extragenic suppressor mutant of gai, called gar2 (gai suppressor 2), was genetically characterized (35).
A third class of gibberellin-signaling mutants is represented by rga (repressor of ga1-3) (40). Rga mutants were found by screening for the ability to suppress the phenotype of the ga1-3 mutant that is impaired in the first step of the gibberellin biosynthesis pathway (41). It is important to note, however, that the rga mutation does not suppress the gibberellin deficiency entirely, because it has no effect on seed germination or on silique growth. All rga alleles are recessive, indicating that RGA most probably encodes a negative regulator of gibberellin signaling.
The RGA gene was cloned using genomic subtraction and shown to be identical to GRS (38). The predicted protein is a member of the VHIID family of plant transcription factors described above. Although primarily repressing the gibberellin response, RGA may also play a role in the control of gibberellin biosynthesis (38). The RGA protein contains the -Asp-Glu-Leu-Leu-Ala-sequence and the Ser/Thr-rich region, both specific to proteins controlling the gibberellin response.
A final regulator, though difficult to place in the pathway at this point, is pickle (pkl). The pkl mutants present a phenotype reminiscent of gibberellin-deficient or gibberellin response mutants and exhibit abnormal root development (42). The primary root meristem of these mutants retained characteristics of embryonic tissues. This phenotype was partially suppressed by addition of gibberellin. Because the gai pkl double mutant exhibited a strong synergistic gibberellin-deficient phenotype, it was concluded that pkl is defective in the gibberellin-response pathway. The gene product PKL is proposed to be a positive regulator of gibberellin signaling (43).
Figure 2 presents a model for gibberellin signal transduction based on currently available genetic and biochemical data (38, 40, 43). Gibberellin interacts with its receptor(s), thereby activating gibberellin responses as a result of inhibition of two negative regulators (branches A and B). In branch A, SPY is acting downstream of (or modulates) GAI, because the spy mutant is completely epistatic to the gai mutation. SPY presumably activates the GAI repressor function via glycosylation. The RGA protein exerts the same function as GAI and represses the transcription of genes involved in gibberellin-regulated growth and development, or it promotes the expression of such a repressor. The repressor function of RGA is activated by SPY or a putative SPY-like protein, unknown to date. This assumption is based on the fact that the rga and spy mutations have an additive effect for the common processes controlled by gibberellins, thus indicating that SPY probably does not regulate RGA. Both O-glycosyltransferases would repress the common gibberellin response (stem elongation, trichome initiation, apical dominance, and flower development). In addition, the "GAI/SPY" branch blocks seed germination and silique growth.
Figure 2. Gibberellin signal transduction. The active gibberellin interacts with a membrane-associated or cytosolic receptor and activates the normal gibberellin response by blocking two repressors, named GAI (branch A) and RGA (branch B). The SPY and/or SPY-like proteins regulate the GAI and RGA proteins via glycosylation. Arrows and bars denote activation and repression, respectively.
Due to their nature, SPY/RGA/GAI would act fairly late in the pathway to induce gibberellin-regulated responses. It should be mentioned that recent evidence also suggests the involvement of heterotrimeric G proteins and cyclic GMP in gibberellin signaling in barley aleurone cells (44, 45). The former intermediates would play a role in the early phase of signal transfer.
4. Downstream Targets
Gibberellin control of plant growth and development is achieved by modulating the expression of specific target genes. One of the best-characterized roles of gibberellins consists in the stimulation of production of hydrolytic enzymes by the aleurone cells. These hydrolytic enzymes, such as a-amylase, a-glucosidase, and a thiol proteinase, are responsible for the breakdown of starch and proteins in the endosperm (46-48). The products of these hydrolyses are absorbed by the scutellum and used by the growing embryo. De novo synthesis of hydrolytic enzymes induced by gibberellin is often due to the presence of a gibberellin response element (GARE) in the promoter region of the corresponding genes (49). The identification of the GARE element has led to the isolation of a myb-like trans-acting factor that can bind to this region, which is closely related to c-Myb and v-Myb consensus sequences (50). Furthermore, gibberellins can stimulate stem elongation by increasing the xyloglucan endotransglycosylase activity, an enzyme that cleaves the rigid structure of the cell wall and is associated with growing tissues (51, 52). In addition, gibberellins cause microtubule reorientation favoring axial elongation (53). The tonoplast-intrinsic protein g-TIP, which maintains turgor pressure, thereby favoring wall extensibility, is also induced by gibberellin application (54). In Petunia flower development, gibberellin induces the expression of genes such as those of chalcone synthase, chalcone isomerase, anthocyanidin synthase, and dihydroflavonol 4-reductase, which are responsible collectively for corolla pigmentation (55-59).
5. Effects
Biologically active gibberellins are involved in key processes of plant growth and development. The best-characterized roles of gibberellins consist in the control of reserve mobilization in cereals following germination and in promotion of shoot elongation through effects on both cell division and expansion. Gibberellins are also involved in the regulation of flower and fruit development and are required for germination and seedling growth in several species (60).
