Gibberellins PDF

Gibberellins  Gibberellins were identified as fungal compounds that promote stem elongation Infection of rice plants with the fungus Gibberella fujikuroi caused the infected plants to elongate rapidly, and therefore unable to support themselves; they are also male sterile. The disease was known as...

Gibberellins  Gibberellins were identified as fungal compounds that promote stem elongation Infection of rice plants with the fungus Gibberella fujikuroi caused the infected plants to elongate rapidly, and therefore unable to support themselves; they are also male sterile. The disease was known as Bakanae disease (Bakanae disease means “foolish seedling”) Uninfected plant Infected, hyper- elongated plants Gibberellins were subsequently identified as endogenous plant growth regulators with multiple roles They stimulate plant growth and developmental transitions Seed germination - hydrolysis of starch reserves for growth Organ Growth – cell expansion (in stem/root, leaves, fruits) Control of flowering - promotion in Arabidopsis, inhibition in mango Fruit set - promotion of ovary growth after fertilization) Induction of parthenocarpy Promotion of sex determination in some species Susceptibility to some abiotic stresses (drought, salt) Gibberellin regulation played an important role during the Green Revolution in development of semi-dwarf hybrids Distinguished plant breeder and Nobel Laureate Norman Borlaug 1914-2009 Semi dwarf varieties are short/sturdy and therefore more resistant to lodging. These invest less in vegetative growth and more into grain production. Many semi-dwarf grain varieties were deficient in GA biosynthesis or response. Many of the pea mutants used by Mendel for study were GA mutants Wild-type pea na, le, lh, sln, la, cry Mendel’s short peas are deficient in GA synthesis.  Gibberellins are a family of tetracyclic diterpenoid compounds More than 100 GAs with various modifications are present in plants. Only a few GAs are biologically active. GA4 is the major active GA in Arabidopsis Sun T (2008) Gibberellin metabolism, perception and signaling pathways in Arabidopsis: September 24, 2008. The Arabidopsis Book. Rockville, MD: American Society of Plant Biologists. doi: 10.1199/tab.0103  The GA biosynthetic pathway is complex and occurs at three places proplastids endomembranes cytoplasm Precursor: Geranyl geranyl pyrophosphate (GGPP) – a 20 carbon compound (also a precursor for carotenoids, chlorophylls, tocopherols and diterpenes/diterpenoids) ent kaurene synthase Stage 3 - cytoplasm KS ent- GGPP CPP GA 13-hydroxylase CPS kaurene copalyl diphosphate synthase GA12 GA53 GA 20-oxidase Stage 1 - proplastid ent-kaurene GA9 GA20 Uniconazole/ ent kaurene oxidase (KO) GA 3-oxidase pacloburazol ent-kaurenoic acid GA4 GA1 ent kaurenoic acid oxidase (KAO) Active GAs GA12 Stage 2 - endomembranes Uniconazole and Paclobutrazol are effective inhibitors of the GA biosynthesis and used for studies of GA roles (inhibit P450 enzymes) The GA biosynthetic pathway in higher plants is complex Active Inactivation Olszewski, N., Sun, T.p., and Gubler, F. (2002) Gibberellin Signaling: Biosynthesis, Catabolism, and Response Pathways Plant Cell 14: S61-S80  Stage 3 - cytoplasm GA 13-hydroxylase GA12 GA53 GA 20-oxidase GA9 GA20 GA 3-oxidase GA4 GA1 Active GAs Nomura et al (2013) Functional Analysis of Arabidopsis CYP714A1 and CYP714A2 Reveals That They are Distinct Gibberellin Modification Enzymes Plant Cell Physiol. 54(11): 1837–1851 (2013)  Loss-of-function mutants of CPS or KS are severely dwarfed due to reduced GA ent kaurene synthase GGPP CPP KS ent- CPS kaurene Wild-type and dwarf CPS copalyl diphosphate KS synthase mutant rice Why does loss of CPS or KS lead to reduced GA? oscps-1 osks-1 WT oscps-1 osks-1 Sakamoto, T., et al. (2004). An overview of gibberellin metabolism enzyme genes and their related mutants in rice. Plant Physiol. 134: 1642-1653.  Loss-of-function mutants of CPS or KS are severely dwarfed due to reduced GA ent kaurene synthase GGPP CPP KS ent- CPS kaurene Wild-type and dwarf CPS copalyl diphosphate KS synthase mutant rice CPS, KS and KAO are encoded by one gene in most plants, so mutants oscps-1 osks-1 have severely reduced GA levels. WT oscps-1 osks-1 Sakamoto, T., et al. (2004). An overview of gibberellin metabolism enzyme genes and their related mutants in rice. Plant Physiol. 134: 1642-1653. Similarly, loss-of-function mutants of KO or KAO are severely dwarfed The pea gene LH encodes ent-kaurene The pea gene NA oxidase (KO) encodes ent- kaurenoic acid oxidase (KAO) ent- kaurene KO ent-kaureonic acid KAO GA12 Davidson, S.E., Elliott, R.C., Helliwell, C.A., Poole, A.T., and Reid, J.B. (2003). The pea gene NA encodes ent-kaurenoic acid oxidase. Plant Physiol. 131: 335-344. Davidson, S.E., Smith, J.J., Helliwell, C.A., Poole, A.T., and Reid, J.B. (2004). The pea gene LH encodes ent-kaurene oxidase. Plant Physiol. 134: 1123-1134.  The GA oxidases are encoded by multiple genes with differing expression patterns GA 13- hydroxylase GA12 GA53 The “green revolution” GA 20- oxidase semidwarf1 rice variety is mutated in a GA20OX that is GA9 GA20 GA 3-oxidase expressed in shoots but not reproductive tissues, leading to GA4 Active GA1 GAs shorter internodes and greater investment of carbon into grain. Imaage courtesy of M. Matsuoka. Sasaki, A., Ashikari, M., Ueguchi-Tanaka, M., Itoh, H., Nishimura, A., Swapan, D., Ishiyama, K., Saito, T., Kobayashi, M., Khush, G. S., Kitano, H. and Matsuoka, M. (2002) A mutant gibberellin-synthesis gene in rice. Nature 416, (6882) 701-702. Similarly, mutants in GA3OX genes have reduced height but not necessarily reduced seed production Pea Arabidopsis GA 13- hydroxylase GA12 GA53 GA 20- oxidase The le mutant that WT Mendel studied is GA9 GA20 mutated in a GA3- GA 3-oxidase oxidase-encoding (le) WT le ga4 gene, as is the ga4 GA4 (ga4) GA1 mutant of Arabidopsis. Lester, D.R., Ross, J.J., Davies, P.J., and Reid, J.B. (1997) Mendel's stem length gene (Le) encodes a gibberellin 3[beta]-hydroxylase. Plant Cell 9: 1435-1443. Chiang, H.H., Hwang, I., and Goodman, H.M. (1995). Isolation of the Arabidopsis GA4 Locus. Plant Cell 7: 195-201.  Paclobutrazol, a GA biosynthesis inhibitor (inhibits kaurene oxidase), can be used for studies on the role of GAs in development 0 M PAC 1 M PAC 5 M PAC 25 M PAC Paclobutrazol is often sprayed on plants to interfere with GA synthesis and prevent branch growth (e.g. in fruit trees). Arabidopsis plants treated with increasing amounts of paclobutrazol (PAC). Adapted from Rieu, I., et al. (2008). Genetic analysis reveals that C19-GA 2-Oxidation is a major gibberellin inactivation pathway in Arabidopsis. Plant Cell 20: 2420-2436. How are the various GAs metabolized after their function?  GAs can be inactivated by several different enzymes HO GA4 GA2 oxidase HO HO cytP450 monooxygenase GA Methyl- (Elongated Uppermost Transferase Internode ) (EUI) (GAMT) HO HO CH3 All modifications are irreversible Over-expression of these enzymes reduces the levels of active GAs Over-expression of the GA deactivating enzyme GA 2- oxidase confers dwarfism due to reduced GA GA 13- Arabidopsis Arabidopsis hydroxylase GA 2- GA12 GA53 GA 2- oxidase oxidase GA 20- oxidase Inactive Inactive forms GA9 GA20 forms GA 3-oxidase GA4 GA1 GA 2-oxidase WT WT GA2ox GA2ox GA2ox GA2ox Inactive Inactive Tobacco forms forms Different GA2oxs can preferentially act on active GAs or their inactive precursors, with WT GA2ox GA2ox different developmental outcomes. Schomburg, F.M., Bizzell, C.M., Lee, D.J., Zeevaart, J.A.D., and Amasino, R.M. (2003). Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants. Plant Cell 15: 151-163. Over-expression of a GA- methyltransferase (GAMT) causes dwarfism GAMT1 GAMT1 Tobacco WT WT Petunia Arabidopsis Varbanova, M., et al. (2007). Methylation of gibberellins by Arabidopsis GAMT1 and GAMT2. Plant Cell 19: 32-45. ELONGATED UPPERMOST INTERNODE encodes a GA-deactivating enzyme (a cytP450 monooxygenase that epoxidizes GAs) Uppermost internode in eui The loss-of-function eui mutant is elongated, Over-expression lines are dwarfed. Zhu, Y., et al. (2006). ELONGATED UPPERMOST INTERNODE encodes a cytochrome P450 monooxygenase that epoxidizes gibberellins in a novel deactivation reaction in rice. Plant Cell 18: 442-456. GA pathway in pea (mutants studied by Mendel for his experiments) Genes of Pea mutants (studied by Mendel) LS = ent kaurene synthase A LH = ent kaurene oxidase NA = ent kaurenoic acid oxidase LE = GA3 oxidase SLN = GA2 oxidase WT le-1 WT na sln nasln NAsln WT na SLN encodes a GA2 oxidase which when mutated leads to GA accumulation and slender plants Yaxley et al. (2001) Gibberellin Biosynthesis Mutations and Root Development in Pea. Plant Physiology 125, 627-633.  GA biosynthesis and metabolism is under complex regulatory control by other hormones and the environment  GA movement: GAs are graft-transmissible; they can move long distances WT - d1 In pea, a mutant na shoot is rescued by grafting onto a Na root. Maize seedlings are grafted d1- d1 side-by-side na In maize, GA or a GA- Na precursor moves from the na wild-type plant to d1 and na promotes growth. Proebsting, W.M., et al. (1992). Gibberellin concentration and transport in genetic lines of pea : Effects of grafting. Plant Physiol. 100: 1354-1360; Katsumi, M., et al. (1983). Evidence for the translocation of gibberellin A3 and gibberellin-like substances in grafts between normal, dwarf1 and dwarf5 seedlings of Zea mays L. Plant Cell Physiol. 24: 379-388 Copyright 1983 Japanese Society of Plant Physiologists, with permission. During seed germination in monocot seeds GA moves from embryo to aleurone to activate amylase GA amylase sugars starch Embryo Endosperm Aleurone  GA promotes growth through cell expansion and division GA induces cell division by expression of cell-cycle regulatory proteins called cyclins. Cell expansion Cell division GA promotes elongation by Cell cell wall loosening and expansion stabilizing the orientation of cortical microtubules, which help direct growth. GA promotes growth through cortical microtubule orientation Uniconazole Circumferential or (GA synthesis Uniconazole hoop-like cortical Control inhibitor) + GA microtubules help promote unidirectional, elongation growth. Inhibition of GA biosynthesis disrupts cortical microtubule arrangement while GA restores arrangement. Inada, S. and Shimmen, T. (2000). Regulation of elongation growth by gibberellin in root segments of Lemna minor. Plant Cell Physiol 41: 932-929, by permission of the Japanese Society of Plant Physiologists. GA promotes growth and cell elongation How is GA signalling regulated? GA signaling: DELLA proteins as negative regulators  DELLA proteins are negative regulators of GA action (and growth) and prevent GA action in absence of GA  DELLA proteins have an N-terminal DELLA (Asp-Glu-Leu-Leu-Ala) domain and a C-terminal GRAS (GAI, RGA and SCARECROW) domain They belong to the GRAS family of proteins which are nuclear-localized transcriptional regulators that seem to lack a DNA-binding motif but bind growth-promoting TFs and prevent them from functioning DELLA domain GRAS domain GA interaction protein-protein interaction The DELLA domain is essential for GA-mediated interactions while GRAS is necessary for protein-protein interactions  DELLAs act as brakes (inhibitors) of growth DELLAs GA GA inactivates DELLA inhibitors (GA is an inhibitor of an inhibitor) Without the inhibitory DELLAs, the plant can respond to growth-promoting activities.  Plants have varying number of DELLA proteins (1-5) Arabidopsis: 5 DELLA proteins GAI (GA-INSENSITIVE 1) RGA (REPRESSOR OF GA1-3) RGL1, RGL2 and RGL3 (RGA-LIKE1 , 2, and 3) Cereals: only one DELLA protein Rice: SLENDER RICE 1 (SLR1) DELLA domain GRAS domain Barley: SLENDER1 (SLN1), Maize: DWARF8 (D8), GA perception Wheat: REDUCED HEIGHT (RHT) Tomato – one (PROCERA); Pea – two (LA and CRY) Arabidopsis: 5 DELLA proteins: Overlapping and distinct functions RGA, RGL2,RGL3, GAI - suppress seed germination GAI, RGA, RGL1 - repress stem elongation RGA, RGL1, RGL2 - repress floral development Regulation of DELLA proteins Transcriptional: GAI and RGA transcribed in most tissues; RGL1-3 transcribed mainly in seeds, seedlings and flowers Post-translational: 1. through proteasome-mediated degradation of DELLA proteins triggered by GA via interactions with the DELLA motif. Mutations in DELLA or TVHYNP domain affect GA responses 2. Modifications of the DELLA proteins (phosphorylation, fucosylation, SUMO modification, N-acetyl glucosame additon etc).  GA-INSENSITIVE1 and SLENDER1 both encode “DELLA” proteins (but their mutants have opposite phenotypes) Rice WT slr1 Stop codon or frameshift Loss of function * GRAS DELLA GRAS domain not functional, domain No inhibition of growth WT gai Constitutive GA responses DELLA domain deletion Gain of function GRAS DELLA domain absent (only C-terminal portion expressed), hence cannot be degraded by GA DELLA protein constitutively active (sequesters Arabidopsis proteins through GRAS domain), no GA responses Peng, J., and Harberd, N.P. (1993). Derivative alleles of the Arabidopsis Gibberellin-Insensitive (gai) mutation confer a wild-type phenotype. Plant Cell 5: 351-360.; Ikeda, A., et al. (2001). 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 13: 999-1010. Mutations in pea DELLA proteins LA and CRY affect root and shoot growth differently NA- entkaurenoic na acid oxidase NA LA CRY na-1 la CRY/cry-s NA la cry-s LA and CRY – DELLA proteins na-1 LA cry-s na-1 la cry-s LA plays a greater role in root and shoot inhibition while CRY-S plays a role in shoot inhibition but not root inhibition Both genes promote transcription of GA biosynthesis genes (GA3 Ox and GA20OX) but suppress transcription of GA 2 oxidase genes (negative feed back regulation) Weston et al., (2008) The Pea DELLA proteins LA and CRY are important regulators of gibberellin synthesis and root growth. Plant Physiology, 147, 199–205, DELLA-encoding genes have had a great agricultural impact for development of semi-dwarf varieties The green-revolution gene reduced height1 from wheat encodes a mutant DELLA. Wild-type wheat reduced-height1 wheat Reprinted by permission of Macmillan Publishers, Ltd. Peng, J., et al. (1999) 'Green revolution' genes encode mutant gibberellin response modulators. Nature 400: 256-261.  DELLA proteins are signalling hubs that connect growth and defense pathways with the environment - Regulate >300 Transcription factors (TFs) and transcriptional regulators (TRs) Mechanism: 1. Sequestration of TFs and/or TRs thereby preventing their function - Suppress GA and growth (binds/sequesters PIF proteins (TFs) that mediate cell elongation in etiolated hypocotyls) - Suppress brassinosteroid responses by sequestering BZR1 (a TF that activates Br responses) - Activate JA responses by sequestering JAZ proteins (TRs) and releasing MYC TFs that activate JA responses (JAZ proteins are JA inhibitors and bind MYCs and prevent their function) - Suppress hook formation by binding EIN3 and preventing its function 2. Enabling function through co-activation of TFs and/or TRs DELLA proteins may also bind certain TFs and help in activation of target genes - Promote seed dormancy by binding ABI3 and ABI5 to enhance target gene expression - Promote photomorphogenesis by activating cytokinin responses through binding of type B ARRs (Arabidopsis Response Regulators). Phokas A and Coates J (2021) Evolution of DELLA function and signaling in land plants. Evolution and development 23:137–154  The PIF3 and PIF4 (bHLH) transcription factors are necessary for post-germinative growth during etiolation (skotomorphogenic growth) PIF3/4 PIF3 and PIF4 activate the transcription of growth- promoting genes, leading to elongation of the hypocotyl in the dark. Reprinted from Davière, J.-M., de Lucas, M., and Prat, S. (2008) Transcription factor interaction: a central step in DELLA function. Curr. Opin. Genet. Devel. 18: 295–303.with permission from Elsevier DELLA proteins bind PIF3 and PIF4 and interfere with their action PIF3/4 DELLA PIF3/PIF4 unable to function in light, Seedlings short Reprinted from Davière, J.-M., de Lucas, M., and Prat, S. (2008) Transcription factor interaction: a central step in DELLA function. Curr. Opin. Genet. Devel. 18: 295–303.with permission from Elsevier GA promotes DELLA degradation, allowing PIF3 and PIF4 to act (in dark) GA DELLA PIF3/4 Reprinted from Davière, J.-M., de Lucas, M., and Prat, S. (2008) Transcription factor interaction: a central step in DELLA function. Curr. Opin. Genet. Devel. 18: 295–303.with permission from Elsevier  DELLAs regulate multiple hormone pathways DELLAs promote dormancy Inhibit cell elongation Inhibit apical hook devp Inhibit trichome devp Inhibit sesquiterpene biosynth Promote JA in defense Promote shoot branching by inhibiting strigolactone action Promote photomorphogenesis through CK Daviere J-M and Achard P (2016) A Pivotal Role of DELLAs in Regulating Multiple Hormone Signals Mol Plant 9: 10-20 Post translational regulation of DELLA proteins DELLA proteins undergo several post-translational modifications that affect their stability and interaction with other proteins and the proteasome machinery - Phosphorylation stabilizes DELLA - Dephosphorylation promotes degradation - SUMO (Small Ubiquitin-like Modifier) modification promotes DELLA interaction with GID1 (GA receptor) in a GA-independent manner, thereby suppressing growth in salt stress - Fucosylation activates DELLA (promotes binding to PIF3, PIF4 and BZR1 and suppresses elongation) - N-acetyl glucosamine attachment reduces DELLA activity (prevents binding to PIFs and BZR1 and allows them to activate elongation) How is GA perceived? How is GA perceived? GA receptors are soluble receptors belonging to the serine hydrolase family, (includes esterases, lipases, and proteases) No enzymatic function detected First identified in rice as gid1 mutant (ga insensitive dwarf 1) GID1 (GA INSENSITIVE DWARF 1) - a single GID1 receptor present in rice gid1 gid1 Ueguchi-Tanaka et al (2005) GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin.Nature 437:693-698 GIBBERELLIN INSENSITIVE DWARF1 (GID1) encodes a GA receptor Unlike biosynthesis mutants, the rice gid1 mutant is not rescued by GA – it is gibberellin-insensitive. WT gid1-1 Reprinted by permission from Macmillan Publishers, Ltd: NATURE. Ueguchi-Tanaka, M., et al. (2005) GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin. Nature 437: 693-698, copyright 2005.  Overexpression of GID1 makes plants hypersensitive to GA GID1 over-expressors bind more GA at a given GA concentration than control and show more GA effects Reprinted by permission from Macmillan Publishers, Ltd: NATURE. Ueguchi-Tanaka, M., et al. (2005) GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin. Nature 437: 693-698, copyright 2005.  Arabidopsis has 3 redundantly-acting GID1 genes Triple mutant WT gid1a-1 gid1b-1 gid1c-1 gid1a-1 gid1b-1 Single mutants gid1c-1 Show functional redundancy (single mutants do not show much of a phenotype). Double mutants show normal flowering responses. gid1a gid1b gid1c triple mutants are severely dwarfed and show no GA responses. Tomato also has three GID receptors Griffiths, J. et al. (2006) Genetic characterization and functional analysis of the GID1 gibberellin receptors in Arabidopsis. Plant Cell18: 3399–3414. GID1 expression is negatively regulated by GA GA downregulates expression of genes involved in its own synthesis (e.g GA3ox) and response (e.g. GID1a,b,c); these are important negative feedback controls. GA3ox1 GA Response GID1 Griffiths, J. et al. (2006) Genetic characterization and functional analysis of the GID1 gibberellin receptors in Arabidopsis. Plant Cell18: 3399–3414. How does GA mediate signaling and responses? When bound to GA, GID1 binds to DELLAs GA-GID-DELLA complex induces degradation of DELLA proteins Reprinted by permission from Macmillan Publishers, Ltd: Nature. Murase, K., Hirano, Y., Sun, T.-p., and Hakoshima, T. (2008). Gibberellin-induced DELLA recognition by the gibberellin receptor GID1. Nature 456: 459-463, Copyright 2008.  GA induces DELLA protein degradation GFP-RGA The RGA DELLA protein is specifically degraded in the presence of GA. Paclobutrazol (PAC) inhibits GA accumulation and indirectly stabilizes RGA. DELLA protein degradation occurs via proteasome Silverstone, A.L., Jung, H.-S., Dill, A., Kawaide, H., Kamiya, Y., and Sun, T.-p. (2001). Repressing a repressor: Gibberellin-induced rapid reduction of the RGA protein in Arabidopsis. Plant Cell 13: 1555-1566.  F-box proteins SLEEPY (SLY) in Arabidopsis and GID2 in rice recognize and bind DELLA proteins Ubiquitin DELLAs DELLAs SLEEPY/GID2 SKP1 E2 RBX1 CUL1 The sleepy and gid2 mutants show reduced growth In absence of SLEEPY and GID2, DELLA is stabilized and GA-induced elongation is inhibited. sleepy1-10 WT gid2-1 WT McGinnis, K.M., Thomas, S.G., Soule, J.D., Strader, L.C., Zale, J.M., Sun, T.-p., and Steber, C.M. (2003). The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase. Plant Cell 15: 1120-1130, From Sasaki, A., et al. (2003) Accumulation of phosphorylated repressor for gibberellin signaling in an F-box mutant. Science 299: 1896-1898, reprinted with permission from AAAS.  SLEEPY (SLY) is required for GA- mediated proteolysis of DELLA proteins WT GA deficient SLEEPY inactive sleepy (sly) mutants do not degrade the RGA DELLA protein in response to GA DELLA accumulates regardless of the presence or absence of GA McGinnis, K.M., Thomas, S.G., Soule, J.D., Strader, L.C., Zale, J.M., Sun, T.-p., and Steber, C.M. (2003). The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase. Plant Cell 15: 1120-1130. slr1 (slender rice1) encodes a mutated non-functional DELLA protein Mutants are elongated and slender due to enhanced GA responses gid2 encodes a mutated non-functional F-box protein required for degradation of DELLAs Mutants are short, dwarf and inhibited in GA responses How would a slr1gid2 double mutant look like? gid2 slr1 The slr1gid2 double mutant looks like slr1 In wild-type rice: DELLA +GA SCFGID2 (SLR1) slr1 mutant gid2 mutant GA response Loss-of-function of SLR1 causes a constitutive GA response, whether or not GID2 is present. WT gid2-1 gid2-1 slr1-1 slr1-1 From Sasaki, A., et al. (2003) Accumulation of phosphorylated repressor for gibberellin signaling in an F-box mutant. Science 299: 1896-1898, reprinted with permission from AAAS. In Arabidopsis, the sleepy mutant, lacking F-box protein is small (because of stabilized DELLA) But if some DELLAs are missing (in a sly1 rga gai background) plants become taller In wild-type Arabidopsis: DELLA +GA SCFSLY (RGA, GAI) Response sly1-10 sly1-10 rga-24 WT gai-t6 Dill, A., Thomas, S.G., Hu, J., Steber, C.M., and Sun, T.-p. (2004). The Arabidopsis F-Box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation. Plant Cell 16: 1392-1405.  GA signaling pathway Low GA High GA GA triggers DELLA protein Growth GA-responsive promotion transcription proteolysis. factor GA GA-responsive transcription DELLA protein factor DELLA Transcription No GA receptor transcription DELLA proteins inhibit growth, in part through blocking transcription. Proteolysis Reprinted from Davière, J.-M., de Lucas, M., and Prat, S. (2008) Transcription factor interaction: a central step in DELLA function. Curr. Opin. Genet. Devel. 18: 295–303.with permission from Elsevier The mutated wheat rht1 allele encodes a REDUCED HEIGHT1 DELLA protein that lacks the DELLA domain and resists proteolysis. Hence constitutive GA suppression and reduced height. Wild-type GA GA-responsive transcription Dwarf rht1 factor GA-responsive RHT1 transcription DELLA factor Transcription rht1 GA rht1 Reprinted by permission of Macmillan Publishers, Ltd. Peng, J., et al. (1999) 'Green revolution' genes encode mutant gibberellin response modulators. Nature 400: 256-261. DELLA proteins are also regulated by processes other than proteolysis The spindly (spy) mutant has an enhanced GA response. SPINDLY (SPY) encodes an N- fucosyl transferase. Addition of fucosyl residue activates DELLA action. spy mutants have inactive DELLA SECRET AGENT (SEC) encodes and N-acetyl glucosamine transferase that inactivates DELLA Daviere J-M and Achard P (2016) A Pivotal Role of DELLAs in Regulating Multiple Hormone Signals Mol Plant 9: 10-20 Zentella, R. et al., (2016) O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis. Genes Dev. 30: 164–176. Zentella, R., et al. (2017) The Arabidopsis O-fucosyltransferase SPINDLY activates nuclear growth repressor DELLA. Nat. Chem. Biol. 13: 479–485 The growth-inhibitory function of DELLAs is a recent evolutionary acquisition This function is exploited in seed plants in the control of seed germination, as well as stress responses. Reprinted from Yasumura, Y., Crumpton-Taylor, M., Fuentes, S., Harberd, N.P. (2007). Step-by-step acquisition of the gibberellin- DELLA growth-regulatory mechanism during land-plant evolution. Curr. Biol. 17: 1225–1230 with permission from Elsevier. GA and ABA act antagonistically in the control of seed germination ABA promotes Germination desiccation tolerance and dormancy Seed germination requires Reserve mobilization elimination of ABA and production of GA to promote growth and breakdown of Cell expansion seed storage products. GA ABA  GA induces expression of nutrient- mobilizing enzymes Breakdown of starch in the endosperm is initiated by GA produced in the embryo GA also activates weakening of the seed coat and aids in its rupture by activating wall hydrolases like XTH and expansins GA amylase DELLA GA BARLEY sugars starch GAMYB Embryo Endosperm Aleurone -amylase GAMYB transcription factors activate amylase Images by Prof. Dr. Otto Wilhelm Thomé Flora von Deutschland, Österreich und der Schweiz 1885 and Chrisdesign.  DELLA stability is crucial to seed dormancy and germination Inhibitory factors Promoting factors ABA Light RGL2 (DELLA) Cold stratification ABI5 (TF) GA PIL5 (TF) Recent studies are elucidating how SPT (TF) these diverse signals form a regulatory network to control the timing of germination Oh, E,, et al. (2007). PIL5, a phytochrome-interacting bHLH protein, regulates gibberellin responsiveness by binding directly to the GAI and RGA promoters in Arabidopsis seeds. Plant Cell 19: 1192-1208. Inhibition of seed germination by ABA is mediated by stabilization of DELLA ABA inhibition of germination requires DELLAs Reprinted from Penfield, S., Gilday, A.D., Halliday, K.J., and Graham, I.A. (2006). DELLA-mediated cotyledon expansion breaks coat-imposed seed dormancy. Curr. Biol. 16: 2366-2370 with permission from Elsevier Copyright 2006. The DELLA protein RGL2 promotes ABA synthesis and signaling RGL2 is a negative regulator of germination that promotes ABA GA synthesis and activation of the ABI5 transcription factor. RGL2 WT ABA rgl2 ABI5 rgl2 Germination ABI5-OX ABI5 is downstream of RGL2 Piskurewicz, U., Jikumaru, Y., Kinoshita, N., Nambara, E., Kamiya, Y., and Lopez-Molina, L. (2008). The gibberellic acid signaling repressor RGL2 inhibits Arabidopsis seed germination by stimulating abscisic acid synthesis and ABI5 activity. Plant Cell 20: 2729-2745.  PIL5 is a TF that inhibits germination PIL5 inhibition of germination requires DELLA Col-0 PIL5OX (DELLA) PIL5 inhibition of germination requires DELLA because the inhibition is reduced in an rga mutant background with reduced DELLA activity. PIL5 RGA RGA functions downstream of PIL5 Oh, E., et al. (2007). PIL5, a phytochrome-interacting bHLH protein, regulates gibberellin responsiveness by binding directly to the GAI and RGA promoters in Arabidopsis seeds. Plant Cell 19: 1192-1208.  GA promotes elongation and submergence avoidance in rice Control Submerged As water levels rise during seasonal Two days of flooding, GA promotes rapid stem submergence induces elongation to keep the top of the tremendous plant above the water line. internode elongation. Kende, H., van der Knaap, E., and Cho, H.-T. (1998). Deepwater rice: A model plant to study stem elongation. Plant Physiol. 118: 1105-1110. Sometimes it is best to limit growth under stressful conditions Slow water rise – avoid submergence by GA-induced growth Rapid water rise – conserve energy by DELLA-mediated growth-inhibition Rice that carry the Sub1A gene can survive flooding by not growing and stabilizing DELLA Control Sub1A Control Sub1A Flooding Fukao, T. and Bailey-Serres , J. (2008). Submergence tolerance conferred by Sub1A is mediated by SLR1 and SLRL1 restriction of gibberellin responses in rice. Proc Natl Acad Sci USA 105: 16814-16819 Copyright © 2008 by the National Academy of Sciences. Under salt stress, survival is enhanced by reducing growth by reduced GA response 170 mM NaCl GA treatment decreases survival in wild-type plants grown on NaCl. Salt stress Magome, H., S. Yamaguchi, et al. (2004). dwarf and delayed-flowering 1, a novel Arabidopsis mutant deficient in gibberellin biosynthesis because of overexpression of a putative AP2 transcription factor. Plant J. 37: 720-729. Under salt stress, survival is enhanced by reduced GA synthesis HighHigh GA2GA2 Ox ox DDF1 activates GA2 ox (degrades GA) (increases survival) GA-deficient plants survive better GA biosynthesis mutant. Magome, H., S. Yamaguchi, et al. (2004). dwarf and delayed-flowering 1, a novel Arabidopsis mutant deficient in gibberellin biosynthesis because of overexpression of a putative AP2 transcription factor. Plant J. 37: 720-729.  At high salt levels, DELLA stabilization and activity is necessary for survival 200 mM salt DELLA loss-of-function mutants – decreased survival GA biosynthesis mutant – enhanced survival DELLA gain-of-function mutant – enhanced survival Achard, P., et al. (2006). Integration of plant responses to environmentally activated phytohormonal signals. Science 311: 91-94; reprinted with permission of AAAS. Ethylene increases salt tolerance through DELLAs 200 mM NaCl Ethylene insensitive mutants have reduced salt tolerance Increased ethylene signaling in ctr1-1 DELLA mutations mutants increases reduce the salt salt tolerance tolerance of ctr1-1 mutants Cao, W.-H. et al., (2007) Modulation of ethylene responses affects plant salt-stress responses. Plant Physiol. 143: 707-719.; Achard, P., et al. (2006). Integration of plant responses to environmentally activated phytohormonal signals. Science 311: 91-94; reprinted with permission from AAAS. Growth and environmental stress: DELLAs integrate diverse signals Submergence Sub1A Salt stress Ethylene Cold stress DELLA function Survival via decreased growth Other effects of DELLAs: Activation of ROS scavenging enzymes to prevent ROS injury to stressed plants GA2ox controls GA movement into the shoot meristem OsGA2ox1 mRNA GA4 GA5 It has been shown in Lolium that Before the transition to GA5 (which is resistant to flowering, OsGA2ox1 is deactivation by GA2ox) can move expressed just below the into the meristem despite GA2ox shoot meristem, selectively activity. excluding GA1 and GA4. Sakamoto, T., Kobayashi, M., Itoh, H., Tagiri, A., Kayano, T., Tanaka, H., Iwahori, S., and Matsuoka, M. (2001). Expression of a gibberellin 2-oxidase gene around the shoot apex is related to phase transition in rice. Plant Physiol. 125: 1508-1516. GA4 enters the meristem after GA2ox expression switches off In Lolium, GA5 levels increase rapidly at the shoot after a single inductive long day, whereas GA4 and GA1 levels accumulate later. GA4 GA5 After floral induction, GA2ox expression switches off, allowing GA4 to move into the meristem and promote stem elongation. Days after induction King, R.W., Moritz, T., Evans, L.T., Junttila, O., and Herlt, A.J. (2001). Long-day induction of flowering in Lolium temulentum involves sequential increases in specific gibberellins at the shoot apex. Plant Physiol. 127: 624-632. Tissue-specific hormone modulation can optimize growth and crop yields ACTPRO::GA2OX Increasing GA catabolism everywhere using a constitutive promoter affects seed production. GA3OXPRO::GA2OX Increasing GA catabolism only in internodes produces high-yielding dwarf rice plants. Reprinted by permission from Macmillan Publishers, Ltd: Nature Biotechnology. Hedden, P. (2003) Constructing dwarf rice. Nature Biotech. 21: 873 - 874 , copyright 2003.  GA biosynthesis and deactivation are tightly controlled Temperature and light Active GAs Auxin upregulates GA regulate downregulate GA synthesis. GA3ox synthesis and Auxin upregulate GA deactivation. Most genes are expressed in a cell- specific manner Thomas, S.G., Phillips, A.L., and Hedden, P. (1999). Molecular cloning and functional expression of gibberellin 2- oxidases, multifunctional enzymes involved in gibberellin deactivation. Proc. Natl. Acad. Sci. USA 96: 4698-4703 Copyright 1999 National Academy of Sciences, USA.  Bimolecular fluorescence complementation YFP-N YFP-C The fluorescent protein YFP can be split into two halves, each of which does not fluoresce. Each half is fused to a protein of interested (Protein A and Protein B). Protein B Protein A  Bimolecular fluorescence complementation YFP-N YFP-C fluorescence Molecular interaction between proteins Protein B A and B Protein A If A and B interact they draw the two halves of YFP together, recreating a fluorescent protein. The interaction between GID1 (receptor) and SLR1 (DELLA) is GA-dependent Bright Field DAPI GID1-only stains (negative control) DNA SLR1-only (negative control) GID1 + SLR1 No GA GID1 + SLR1 With GA Ueguchi-Tanaka, M., et al. (2007) Molecular interactions of a soluble gibberellin receptor, GID1, with a rice DELLA protein, SLR1, and gibberellin. Plant Cell 19: 2140-2155. How can you differentiate between a hormone deficient mutant and hormone response mutant  GA responses Biosynthesis mutant Response mutant Response mutant No GA signaling Constitutive GA even when supplied signaling with external GA Harberd, N.P., Belfield, E., and Yasumura, Y. (2009). The angiosperm gibberellin-GID1-DELLA growth regulatory mechanism: How an “inhibitor of an inhibitor" enables flexible response to fluctuating environments. Plant Cell 21: 1328-1339.  GA-INSENSITIVE1 and SLENDER1 both encode “DELLA” proteins WT slr1 Loss of DELLA repressor – GA Response strong constitutive response WT gai Enhanced DELLA GA Response repressor – no response Peng, J., and Harberd, N.P. (1993). Derivative alleles of the Arabidopsis Gibberellin-Insensitive (gai) mutation confer a wild-type phenotype. Plant Cell 5: 351-360.; Ikeda, A., et al. (2001). 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 13: 999-1010. Ubiquitin ligase complexes ubiquitinate target proteins Ubiquitin Target Target F-box SKP1 E2 RBX1 CUL1  Ubiquitinated targets are proteolyzed by the 26S proteasome Target The proteasome breaks down target proteins and recycles ubiquitin. 26S proteasome