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Gibberellin signalling pathway

Kenji Gomi and Makoto Matsuoka
Recent molecular biological and genetical studies have identied several positive and negative regulators of gibberellin (GA) signalling pathways in higher plants. The DELLA protein functions as a negative regulator of GA signalling; its degradation through the ubiquitin/proteasome pathway is a key event in the regulation of GA-stimulated processes.
Addresses BioScience Centre, Nagoya University, Nagoya 464-8601, Japan e-mail: makoto@agr.nagoya-u.ac.jp

an almost complete cascade of the GA biosynthetic process [1]. In contrast to the GA-biosynthesis cascade, the mechanisms of GA signal transduction are still poorly understood. Recent studies have revealed that a negative regulator functions as a molecular switch in GA signalling [610,11]. In this review, we summarise recent ndings on the GA signalling pathway, focusing on studies carried out in the past two years.

Positive regulation of GA signalling

GAMYB is a GA-regulated MYB transcription factor that was rst identied as an activator of a-amylase expression in barley aleurone cells [1214]. A recent study demonstrated that the role of GAMYB is not restricted to aleurone cells but is also involved in anther development in barley [15]. Furthermore, GAMYB interacts with KGM (KINASE-ASSOCIATED WITH GAMYB), which was isolated by yeast two-hybrid screening using GAMYB as bait. KGM is a member of an emerging subgroup of protein kinases and it represses GAMYB function in barley aleurone [16]. Although the phosphorylation of GAMYB by KGM has not been demonstrated and the detailed function of KGM is not yet clear, the characterisation of KGM function will provide new insights into GA signalling pathways. The rice dwarf1 (d1) mutant has a GA-insensitive dwarf phenotype. The D1 gene encodes an a-subunit of heterotrimeric G proteins [17,18]. Phenotypic analysis revealed that increased expression of the GA 20-oxidase gene led to the accumulation of high concentrations of bioactive GA in the stunted internodes of d1 mutants. Furthermore, analysis of a d1 and slender rice1 (slr1) double mutant revealed that SLR1 is epistatic to D1 [19]. These results indicate that D1 functions as a positive regulator of GA signalling. Some recent studies have demonstrated, however, that the D1 protein also functions in auxin signalling and disease resistance [20,21]. More precise analysis will be necessary to identify the overall function of the D1 protein. REPRESSION OF SHOOT GROWTH (RSG) is thought to be a positive regulator of the GA-biosynthetic pathway in tobacco. Transgenic tobacco plants expressing a dominant-negative form of RSG had a dwarf phenotype and a reduced concentration of the active GA, GA1. RSG, which contains a basic leucine-zipper (bZIP) domain, transactivated the expression of the ent-kaurene oxidase gene through interaction with its promoter sequence [22]. Recently, it has been demonstrated that 14-3-3 proteins bind RSG and control its subcellular localisation, thus regulating its efciency as a transcriptional effector of GA-synthesis genes in the nucleus [23].
Current Opinion in Plant Biology 2003, 6:489493

Current Opinion in Plant Biology 2003, 6:489493 This review comes from a themed issue on Cell signalling and gene regulation Edited by Kazuo Shinozaki and Elizabeth Dennis 1369-5266/$ see front matter 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/S1369-5266(03)00079-7

Abbreviations d1 dwarf1 GA gibberellin gai gibberellic-acid insensitive GAMYB GA-regulated MYB transcription factor GFP green uorescent protein gid1 GA-insensitive dwarf1 KGM KINASE ASSOCIATED WITH GAMYB PHOR1 PHOTOPERIOD-RESPONSIVE1 rga repressor of ga13 Rht Reduced height RSG REPRESSION OF SHOOT GROWTH SCF Skp1cullinF-box Skp1 Suppressor of kinetochore protein1 sln1 slender1 slr1 slender rice1 SLY1 SLEEPY1 spy spindly

Introduction
Gibberellins (GAs) are a large family of tetracyclic diterpenoid plant growth regulators. To date, 126 GAs have been identied in higher plants, fungi and bacteria [1]. They are associated with several plant growth and development processes, such as seed germination, stem elongation, owering, fruit development and the regulation of gene expression in the cereal aleurone layer [24]. Many GA-related mutants have been isolated from various plant species [3,4,5], and these mutants have helped to determine the physiological role of GA and to elucidate its biosynthetic pathways. Genes that encode GA-catalytic enzymes have been identied using GA-decient mutants, enabling researchers to build
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490 Cell signalling and gene regulation

PHOTOPERIOD-RESPONSIVE1 (PHOR1) encodes the armadillo-repeat (arm-repeat) protein, which is upregulated in potato leaves under conditions that induce tuberisation. PHOR1-antisense plants have a semi-dwarf phenotype similar to that of GA-decient mutants and exhibit reduced GA responsiveness. A PHOR1::green uorescent protein (GFP) construct was transported from the cytosol into the nucleus in response to GA treatment [24], suggesting that PHOR1 acts as a positive regulator in GA signalling. The GA-insensitive dwarf1 (gid1) rice mutant has a GAinsensitive dwarf phenotype [25]. The GID1 gene encodes a member of the serine hydrolase family, which includes esterases, lipases, and proteases [25,26]. Although the enzymatic function of GID1 has not yet been identied, analysis of a gid1 and slr1 double mutant has revealed that SLR1 is epistatic to GID1. Consistent with the idea that GID1 acts upstream of SLR1, SLR1 was not degraded in the gid1 mutant when treated with GA, whereas GA treatment causes rapid degradation of SLR1 in wildtype plants [26]. Recent studies have indicated that the GID1 protein may be directly involved in the degradation of the SLR1 protein (M Ueguchi-Tanaka, M Matsuoka, unpublished data).

those of rice slr1 and barley sln1 mutants, induced a constitutive GA-responding phenotype [10,11]. These results demonstrate that DELLA proteins function as negative regulators of GA signalling, and that the DELLA and TVHYNP domains are essential for this function. Further domain analysis using transgenic rice plants that overproduced different kinds of truncated SLR1 proteins revealed that the SLR1 protein can be divided into four parts: a GA signal perception domain at the amino terminus (DELLA and TVHYNP), a regulatory domain that controls the proteins repression activity (S/T-rich domain), a dimer-formation domain (leucine zipper), and a repression domain at the carboxyl terminus [11]. DELLA proteins are localised in the nuclei where they suppress the downstream GA action and are rapidly degraded in response to GA signals [2931]. It has been suggested recently that GA-dependent degradation of DELLA proteins is a key event for GA-signalling. Arabidopsis spindly (spy) was rst identied as a mutant that is resistant to the GA-biosynthesis inhibitor paclobutrazol (PAC); spy germinates in the presence of PAC, which blocks the germination of wildtype Arabidopsis [32]. The SPY gene encodes a protein that is homologous to O-GlcNAc transferase. Recent studies on the overexpression of SPY have indicated that SPY functions not only as a negative regulator of GA signalling but also in other signalling pathways. Filardo and Swain [33] discussed the possibilities of the negative regulation of PHOR1 function by SPY and of crosstalk between the GA response and the circadian clock.

Negative regulation of GA signalling

GA-insensitive mutants that are defective in the DELLA genes have been identied in screens of various plant species, such as Arabidopsis (repressor of ga13 [rga] and gibberellic-acid insensitive [gai]), barley (slender1 [sln1]), maize (Dwarf8 [D8]), wheat (Reduced height [Rht]), and rice (slr1) [610,11,27]. These mutants fall into two classes: those caused by semi-dominant gain-of-function mutations in Arabidopsis, maize, barley, and wheat, which lead to GA-insensitive dwarsm; and those caused by recessive loss-of-function mutations in barley and rice, which lead to increased growth. The wheat Rht allele was used to produce the wheat varieties that enabled the green revolution [8]. The DELLA proteins are members of the GRAS family, which also includes SCARECROW and SHORT ROOT [28]. In addition to the GRAS family consensus motifs, GA-signal-related DELLA proteins also contain unique motifs in their amino-terminal region called DELLA domains. These domains are absent from other GRAS proteins. The sequence of the Arabidopsis gai allele demonstrated that in-frame deletion mutations in the DELLA domain induced the GA-insensitive dwarf phenotype of gai mutants [6]. Similarly, wheat Rht-B1/Rht-D1 and maize D8 alleles also have an in-frame deletion in the DELLA or TVHYNP domain, respectively [8]. Furthermore, transgenic rice plants that overproduced an SLR1 protein that had a truncated DELLA domain showed a dominant GA-insensitive dwarf phenotype [10,11]. On the other hand, null alleles for these proteins, such as
Current Opinion in Plant Biology 2003, 6:489493

Involvement of the ubiquitin/proteasome pathway in GA signalling

Very recently, we isolated a new GA-insensitive dwarf rice mutant, gid2, which has a severe dwarf phenotype without any GA-responses [34]. The GID2 gene encodes a putative F-box protein, which interacted with a rice Skp1 (Suppressor of kinetochore protein1) homologue in a yeast two-hybrid assay. GID2 is therefore expected to form a Skp1cullinF-box (SCF) complex and to function as an E3 ubiquitin ligase. In gid2 mutants, SLR1 accumulates in a phosphorylated form even though these mutants also accumulate high levels of active GA. In contrast, SLR1 is rapidly degraded by GA through ubiquitination in the wildtype [34]. The Arabidopsis SLEEPY 1 (SLY1) gene has been isolated by positional cloning and found to encode a putative F-box protein with structural similarity to rice GID2. In the sly1 mutant, the DELLA protein RGA accumulates to high concentrations even after GA treatment [35]. Furthermore, a recent phenotypical analysis of barley sln1 revealed that the degradation of SLN1 protein is inhibited by proteasome and kinase inhibitors [36]. These ndings indicate that GA induces the degradation of DELLA-repressor proteins through the ubiquitin/proteasome pathway, mediated by the SCF complex. The degradation mechanisms are probably not uniform
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Gibberellin signalling Gomi and Matsuoka 491

for all DELLA proteins, however, because Arabidopsis GAI is not degraded as a result of GA3 treatment [37]. Further biochemical studies are needed to solve the overall function of the DELLA proteins in the GA signalling pathway. PHOR1, which contains an U-box domain at its amino terminus, may also be involved in the degradation of DELLA proteins. The U-box motif was rst identied in yeast UFD2, an E4 multiubiquitin chain assembly factor, and was later recognised as a conserved domain that is shared with proteins in various kinds of organisms [38]. In Arabidopsis, 37 predicted U-box proteins have been identied in database searches [39], although their biological function has not yet been characterised. The predicted three-dimensional structure of the U-box domain is similar to that of RING ngers [40], some of which have E3 ubiquitin ligase activity in triggering the
Figure 1

degradation of target proteins [41]. Recent studies have demonstrated that U-box proteins in mammals possess ubiquitin ligase activity [42,43]. In this context, PHOR1 may act as an E3 ubiquitin ligase that degrades DELLA proteins in the GA signalling pathway.

Conclusions
The processing of the GA signal in the nucleus depends directly on the presence or absence of DELLA proteins, which are therefore presently considered to be a molecular switch for GA signalling (Figure 1). An SCF complex is essential for the degradation of DELLA proteins, which results in the transduction of the GA signal. Rice GID2 and Arabidopsis SLY1 are F-box proteins, which are typically components of SCF complexes. Many F-box proteins contain a proteinprotein interaction domain, such as a leucine-rich repeat (LRR) or a WD-40 repeat sequence, which allows their interaction with target

GA

GA D1 GA signal PHOR1 Kinase SPY GID1 14-3-3 protein GID2/SLY1 SLR1/RGA KGM PHOR1

RSG

RSG

GAMYB Nucleus GA-response genes

GA-mediated action Cytoplasm

Current Opinion in Plant Biology

Possible roles of recently identified factors in the GA signalling pathway. In the absence of GA, DELLA protein (SLR1/RGA) directly or indirectly inhibits the expression of GA-induced genes, including GAMYB. KGM inhibits GAMYB activity by phosphorylation. SPY activates the negative regulator and inhibits the activity of the positive regulator by O-GlcNAc modification. GA binds to an unidentified GA receptor(s) and activates G proteins (D1) that enhance the GA signal. PHOR1 is translocated into the nucleus, where it acts as a positive regulator by GA signalling. The GA signal also activates protein kinase and GID1 to trigger GID2/SLY1-mediated degradation of SLR1/RGA. 14-3-3 proteins regulate the subcellular localisation of RSG, which controls the expression of the ent-kaurene oxidase gene. www.current-opinion.com Current Opinion in Plant Biology 2003, 6:489493

492 Cell signalling and gene regulation

proteins [4446]. GID2 and SLY1 do not contain such interactive sequences, however, suggesting that they may not interact directly with SLR1 and RGA. Additional protein(s) may be required as mediators of the interaction between GID2/SLY1 and their target proteins, SLR1/ RGA, respectively. Phosphorylation of the DELLA protein (SLR1) triggers its ubiquitin/proteasome-mediated degradation through interaction with SCFGID2. In barley, a tyrosine kinase inhibitor blocked the GA-induced degradation of SLN1 [36], indicating that GA-dependent phosphorylation of DELLA protein forms part of the GA signalling pathway. Thus, the next steps in unravelling the GA signalling pathway are to identify DELLA-protein-specic kinases and the phosphorylation site of DELLA proteins. Furthermore, as DELLA proteins may not contain DNA-binding domain, even though they seem to function as trans-acting factors, other interacting factors that contain DNA-binding motifs should exist. At present, there is little information on the relationship between GAMYB and DELLA proteins. GA-induced expression of GAMYB was inhibited in the barley dominant Sln1 mutant, indicating that SLN1 functions upstream of the transcription of GAMYB in barley aleurone cells [31,47]. Whether GAMYB and DELLA proteins interact directly is unclear; other protein(s) may be necessary to transduce the signal from a DELLA protein to GAMYB. Finally, the nature of the primary GA receptor is still obscure, despite great efforts to identify it. At present, favoured hypotheses assume that there may be two types of GA receptor in plant cells; one a plasma-membrane-bound receptor and the other a cytoplasm-type receptor. The isolation and characterisation of the GA receptor is one of the most important targets in this eld.

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10. Ikeda A, Ueguchi-Tanaka M, Sonoda Y, Kitano H, Koshioka M, Futsuhara Y, Matsuoka M, Yamaguchi J: Slender rice, a constitutive gibberellin response mutant, is caused by a null mutation of the SLR1 gene, an ortholog of the height-regulating gene GAI/RGA/RHT/D8. Plant Cell 2001, 13:999-1010. 11. Itoh H, Ueguchi-Tanaka M, Sato Y, Ashikari M, Matsuoka M: The  gibberellin signaling pathway is regulated by the appearance and disappearance of SLENDER RICE1 in nuclei. Plant Cell 2002, 14:57-70. This study shows that SLR1 represses the GA signalling pathway in the nucleus in rice, and that the application of GA3 induces the disappearance of nuclear SLR1::GFP protein. By analysis of truncated SLR1::GFP proteins, the authors revealed that the SLR1 protein can be divided into four parts: a GA signal-perception domain, a regulatory domain for its repression domain, a dimer-formation domain that is essential for signal perception and repression activity, and a repression domain. 12. Gubler F, Kalla R, Roberts J, Jacobsen JV: Gibberellin-regulated expression of a myb gene in barley aleurone cells: evidence for Myb transactivation of a high-pI a-amylase gene promotor. Plant Cell 1995, 7:1879-1891. s D, Keys M, Watts R, Mundy J, Jacobsen JV: 13. Gubler F, Ravento Target genes and regulatory domains of the GAMYB transcriptional activator in cereal aleurone. Plant J 1999, 17:1-9. s M, Go mez-Cadenas A, Ho THD: Hormonal regulation of a 14. Cerco cysteine protease gene, EPB-1, in barley aleurone layers: cis- and trans-acting elements involved in the coordinated gene expression regulated by gibberellins and abscisic acid. Plant J 1999, 19:107-118. 15. Murray F, Kalla R, Jacobsen J, Gubler F: A role of HvGAMYB in  anther development. Plant J 2003, 33:481-491. The authors of this paper demonstrate that transgenic barley that overexpresses HvGAMYB shows a decrease in anther size and is male sterile: defects that are associated with the increase in HvGAMYB levels. Application of GA3 increases HvGAMYB level and decreases SLN1 levels in anthers as well as in cereal aleurone. 16. Woodger FJ, Gubler F, Pogson BJ, Jacobsen JV: A Mak-like  kinase is a repressor of GAMYB in barley aleurone. Plant J 2003, 33:707-717. The authors isolated KGM as a GAMYB-binding partner from barley by yeast two-hybrid screening. KGM is a new member of the emerging malegerm-cell-associated (Mak)-subgroup of cdc2-related and mitogen-activated protein (MAP) kinase-related protein kinases. Transient expression assays were used to show that KGM specically represses a-amylase promoter activity. 17. Ashikari M, Wu J, Yano M, Sasaki T, Yoshimura A: Rice gibberellin-insensitive dwarf mutant gene Dwarf 1 encodes the alpha-subunit of GTP-binding protein. Proc Natl Acad Sci USA 1999, 96:10284-10289. 18. Fujisawa Y, Kato T, Ohki S, Ishikawa A, Kitano H, Sasaki T, Asahi T, Iwasaki Y: Suppression of the heterotrimeric G protein causes abnormal morphology, including dwarsm, in rice. Proc Natl Acad Sci USA 1999, 96:7575-7580. 19. Ueguchi-Tanaka M, Fujisawa Y, Kobayashi M, Ashikari M, Iwasaki Y, Kitano H, Matsuoka M: Rice dwarf mutant d1, which is defective in the a subunit of the heterotrimeric G protein, affects gibberellin signal transduction. Proc Natl Acad Sci USA 2000, 97:11638-11643. www.current-opinion.com

Acknowledgements
Our research on GA is supported by a Grant-in-Aid for Centres of Excellence, a Grant-in-Aid from the program for the Promotion of Basic Research Activities for Innovative Bioscience, and by the Ministry of Agriculture, Fisheries and Food (MAFF) Rice Genome Project.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. 2. 3. 4. 5.  Hedden P, Phillips AL: Gibberellin metabolism: new insights revealed by the genes. Trends Plant Sci 2000, 5:523-530. Reid JB: Plant hormone mutants. J Plant Growth Regul 1993, 12:207-226. Hooley R: Gibberellin: perception, transduction and responses. Plant Mol Biol 1994, 26:1529-1555. Ross JJ, Murfet IC, Reid JB: Gibberellin mutants. Physiol Plant 1997, 100:550-560.

Olszewski N, Sun TP, Gubler F: Gibberellin signaling: biosynthesis, catabolism, and response pathway. Plant Cell 2002, 14:61-80. This is a readable summary that provides updated and detailed information on the GA biosynthetic and signalling pathway. Current Opinion in Plant Biology 2003, 6:489493

Gibberellin signalling Gomi and Matsuoka 493

20. Fujisawa Y, Kato H, Iwasaki Y: Structure and function of heterotrimeric G proteins in plants. Plant Cell Physiol 2001, 42:789-794. 21. Suharsono U, Fujisawa Y, Kawasaki T, Iwasaki Y, Satoh H, Shimamoto K: The heterotrimeric G protein alpha subunit acts upstream of the small GTPase Rac in disease resistance of rice. Proc Natl Acad Sci USA 2002, 99:13307-13312. 22. Fukazawa J, Sakai T, Ishida S, Yamaguchi I, Kamiya Y, Takahashi Y: REPRESSION OF SHOOT GROWTH, a bZIP transcriptional activator, regulates cell elongation by controlling the level of gibberellins. Plant Cell 2000, 12:901-915. 23. Igarashi D, Ishida S, Fukazawa J, Takahashi Y: 14-3-3 proteins regulate intracellular localization of the bZIP transcriptional activator RSG. Plant Cell 2001, 13:2483-2497. a-Mart 24. Amador V, Monte E, Grac nes JL, Prat S: Gibberellins signal nuclear import of PHOR1, a photoperiod-responsive protein with homology to Drosophila armadillo. Cell 2001, 106:343-354. 25. Sasaki A, Ashikari M, Ueguchi-Tanaka M, Itoh H, Kobayashi M, Kitano H, Matsuoka M: Screening of rice GIBBERELLININSENSITIVE DWARF 1 mutants (GID1). In Proceedings of the 17th International Conference on Plant Growth Substances. July 16 2001; Brno, Czech Republic. 26. Ueguchi-Tanaka M, Ashikari M, Itoh H, Kobayashi M, Kitano H, Matsuoka M: Characterization of rice dwarf mutant, GIBBERELLIN-INSENSITIVE DWARF 1 (GID1). In Proceedings of the 17th International Conference on Plant Growth Substances. July 16 2001; Brno, Czech Republic. 27. Ogawa M, Kusano T, Katsumi M, Sano H: Rice gibberellininsensitive gene homolog, OsGAI, encodes a nuclear-localized protein capable of gene activation at transcriptional level. Gene 2000, 245:21-29. 28. Pysh LD, Wysocka-Diller JW, Camilleri C, Bouchez D, Benfey PN: The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROW-LIKE genes. Plant J 1999, 18:111-119. 29. Dill A, Jung HS, Sun TP: The DELLA motif is essential for gibberellin-induced degradation of RGA. Proc Natl Acad Sci USA 2001, 98:14162-14167. 30. Silverstone AL, Jung HS, Dill A, Kawaide H, Kamiya Y, Sun TS: Repressing a repressor: gibberellin-induced rapid reduction of the RGA protein in Arabidopsis. Plant Cell 2001, 13:1555-1566. 31. Gubler F, Chandler PM, White RG, Llewellyn DJ, Jacobsen JV: Gibberellin signaling in barley aleurone cells. Control of SLN1 and GAMYB expression. Plant Physiol 2002, 129:191-200. 32. Jacobsen SE, Olszewski NE: Mutations at the SPINDLY locus of Arabidopsis alter gibberellin signal transduction. Plant Cell 1993, 5:887-896. 33. Filardo FF, Swain SM: SPYing on GA signaling and plant development. J Plant Growth Regul 2003, in press. 34. Sasaki A, Itoh H, Gomi K, Ueguchi-Tanaka M, Ishiyama K,  Kobayashi M, Jeong DH, An G, Kitano H, Ashikari M, Matsuoka M: Accumulation of the phosphorylated repressor for GA signaling in an F-box mutant. Science 2003, 299:1896-1898.

The authors screened a rice GA-insensitive dwarf mutant, gid2, and isolated the GID2 gene, which encodes a novel F-box protein. In yeast cells, the GID2 protein interacted with the rice homologue of Arabidopsis Skp1, a component of SCF ubiquitin ligases. Phosphorylated SLR1 protein accumulated to high levels in gid2 mutants. The authors proposed that SLR1 is phosphorylated in a GA-dependent manner and is degraded by an SCFGID2 complex through ubiquitination. 35. McGinnis KM, Thomas SG, Soule JD, Strader LC, Zale JM, Sun T-P,  Steber CM: The Arabidopsis SLEEPY1 (SLY1) gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase. Plant Cell 2003, 15:1120-1130. The authors show that the SLY1-gene-encoded F-box protein, together with RGA protein, accumulates to high levels in sly1 mutants. This nding indicates that SCFSLY1 triggers the degradation of RGA in a GA-dependent manner. 36. Fu X, Richards DE, Ait-Ali T, Hynes LW, Ougham H, Peng J,  Harberd NP: Gibberellin-mediated proteasome-dependent degradation of the barley DELLA protein SLN1 repressor. Plant Cell 2002, 14:3191-3200. The authors use specic proteasome inhibitors to demonstrate that the ubiquitin/proteasome pathway is necessary for GA-dependent degradation of SLN1 in barley. Furthermore, they use protein kinase or phosphatase inhibitors to show that protein phosphorylation or dephosphorylation is required for the degradation of SLN1 by the proteasome. 37. Fleck B, Harberd NP: Evidence that the Arabidopsis nuclear gibberellin signaling protein GAI is not destabilized by gibberellin. Plant J 2002, 32:935-947. 38. Koegl M, Hoppe T, Schlenker S, Ulrich HD, Mayer TU, Jentsch S: A novel ubiquitination factor, E4, is involved in multi assembly. Cell 1999, 96:635-644. 39. Azevedo C, Santos-Rosa MJ, Shirasu K: The U-box protein family in plants. Trends Plant Sci 2001, 8:354-358. 40. Aravind L, Koonin EV: The U box is a modied RING nger a common domain in ubiquitination. Curr Biol 2000, 10:132-134. 41. Joazeiro CAP, Weissman AM: RING nger proteins: mediators of ubiquitin ligase activity. Cell 2000, 102:549-552. ller U, Harbers K: Interaction of 42. Pringa E, Martinez-Noel G, Mu the RING nger-related U-box motif of a nuclear dot protein with ubiquitin-conjugating enzymes. J Biol Chem 2001, 276:19617-19623. 43. Hatakeyama S, Yada M, Matsumoto M, Ishida N, Nakayama K: U box proteins as a new family of ubiquitin-protein ligases. J Biol Chem 2001, 276:33111-33120. 44. Deshaies RJ: SCF and Cullin/Ring H2-based ubiquitin ligases. Annu Rev Cell Dev Biol 1999, 15:435-467. 45. Gagne JM, Downes BP, Shiu SH, Durski AM, Vierstra RD: The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis. Proc Natl Acad Sci USA 2002, 99:11519-11524. 46. Kuroda H, Takahashi N, Shimada H, Seki M, Shinozaki K, Matsui M: Classication and expression analysis of Arabidopsis F-boxcontaining protein genes. Plant Cell Physiol 2002, 43:1073-1085. mez-Cadenas A, Zentalla R, Walker-Simmons M, Ho T-HD: 47. Go Gibberellin/abscisic acid antagonism in barley aleurone cells: site of action of protein kinase PKABA1 in relation to gibberellin signaling molecules. Plant Cell 2001, 13:667-679.

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