Indian Journal of Biotechnology Vol 2, 1anuary 2003, pp 9-16

Hormonal Regulation of Development and the Possible Origin of Plant Hormonal Responses in Bryophytes

M M Johri* and Jacinta S D'Souza Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India

The protonema of is a far simpler paradigm to understand the mechanism of hormonal action and tolerance to abiotic stresses in plants. Its developmental biology, responses to hormones and the similarity of signaling mechanisms with higher plants are reviewed. There is strong evidence for second messenger role of calcium ions in the action of cytokinin. Multiple calcium-dependent protein kinases (CDPKs) are present in the protonema. The Funaria hygrometrica CDPK gene (FhCDPK) shows the characteristic catalytic and autoinhibitory domains, the four EF hands and the highest homology to CDPKs from higher plants but far lower to liverwort or other moss CDPK genes. A 38 kDa myelin basic protein kinase (MBP kinase) is activated within minutes by abscisic acid (ABA) and salinity. As ABA also confers tolerance against desiccation and freezing and the wheat ABA-inducible promoter is fully functional in mosses, the ABA signaling mechanism seems to be highly conserved. In plants, the CDPKs are involved not only in hormonal signaling but also in the acclimation response against abiotic stresses. The manipulation of signal transduction components such as transcription factors, CDPKs and calcineurin have emerged as viable strategies to genetically engineer the stress tolerant plants. There is increasing evidence to support the origin of plant hormonal responses at the level of bryophytes.

Keywords : Funaria hygrometrica, moss protonema, CDPKs, MBP kinase, abscisic acid, auxin, calcium, origin of hormonal responses

Introduction 1993; Johri, 1978). The hormonal responses are rapid The overall development in plants is regulated by and discernible either in single cells or in a group of a environmental and internal factors. How plants few cells and are thus more or less cell autonomous. perceive various signals, transduce them and This review focusses on the hormonal regulation of ultimately alter the growth in terms of temporal .and protonema development, the diversity of protein spatial patterns, is beginning to be comprehended. kinases (PKs) from the moss, F. hygrometrica and the The bryophytes represent the earliest group of land importance of PKs in relation to abiotic stress plants where the phytohormone-mediated morpho- tolerance in plants. The possible origin of hormonal genetic responses comparable to that in higher plants responses is also discussed. have been found (Bopp, 1990; Johri, 1990). The protonema of mosses such as Funaria hygrometrica Hormonal Regulation of Cell differentiation in Moss Hedw. and (Hedw.) B.S.G. Suspension Cultures have been used to study the hormonal regulation of A remarkable feature of the protonemal suspension development (Bopp & Atzorn, 1992; Cove & Knight, cultures of Funaria has been the long-term stability of the cell line J-2 which has now been maintained for *Author for correspondence: over 32 years by repeated subculture in a low-calcium Tel: 022-22152971 Ext-2255 ; Fax: 022-22152110 medium. During this long time, it has neither lost the E-mail: [email protected] potential to differentiate nor the responsiveness to Abbreviations: phytohormones (Johri, 1974). The caulonema differen- A23187: calcium ionophore; ABA: abscisic acid; CaM: calmodulin; CCaMK: calcium regulated calmodulin-dependent PK; CDPK: tiation is regulated by inoculum size, auxin concen- calcium-dependent protein kinase; CTC: chlorotetracycline; DHP: tration and nutrient level. It marks a major develop- IA-dihydropyridine; FhCDPK: Funaria hygrometrica CDPK gene; mental switch, which is turned on by biologically LEA: late embryogenesis abundant; MAPKKK: mitogen activated active auxins and ethyl ester of IAA but not by protein kinase kinase kinase; MBP kinase: myelin basic protein indoleacrylic acid or 2,4-dichlorophenoxyacetic acid kinase; PCIB: p-chlorophenoxyisobutyric acid; PK: Protein kinase; SAPK: stress activated PK; SIMK: salt stress-induced MAPK. (Johri & Desai, 1973; Johri & D'Souza, 1990). There is 10 INDIAN J BIOTECHNOL, JANUARY 2003

an increase In endogenous auxin during caulonema The Role of Calcium in Mosses formation (Atzorn et al, 1989a, 1989b). The The role of calcium in cytokinin induced bud cytokinins induce the formation of bud initials, which formation in Funaria and Physcomitrella is well- arise as side branches from caulonemal cells (Gorton documented (Schumaker & Dietrich, 1997). Using the & Eakin, 1957). Cytokinin-over-producing mutants of lipophilic fluorescent calcium chelating probe-- P. patens form the buds constitutively (Ashton et al, chlorotetracycline (CTC), a calcium rise following 1979). cytokinin treatment was localised to the presumptive ABA inhibits the growth and cytokinin-induced initial cell site (Saunders & Hepler, 1981). bud formation (Valdon & Mummery, 1971). ABA- Measurements of calcium currents using vibrating treatment of protonemal cells is reported to increase microelectrodes along a caulonema filament have tolerance against freezing and to confer adaptation to shown that cytokinin application leads to an increase drought (Nagao et al, 2001; Werner et al, 1993). The in the magnitude of the inward current and causes a endogenous level of ABA increases in moss plants change in the spatial properties of the current under arid conditions (Bopp & Werner, 1993) and an (Saunders, 1986). In a caulonemal cell maximum application of ABA causes closure of stomata in the inward current is observed near the nucleus but after Funaria sporophyte (Garner & Paolillo, 1973). In the cytokinin application, there is a decrease in the presence of ABA, the side branches on the protonema current near the centre which is followed by a rise in remain short and develop into resting structures the apical end. This rise in the inward current predicts referred to as the brood cells (Johri, 1988). Formation the location of the initial cell and falls to resting levels of new polypeptides which share epitopes with the with the onset of the initial cell outgrowth. This higher plant LEA (late embryogenesis abundant) current can be blocked by gadolinium, which proteins, have been demonstrated in the protonema competitively inhibits calcium transport. (Ainapure, 1998). The ABA- and osmotic stress- The calcium channel blockers, verapamil and inducible promoter elements from the wheat Em gene D-600 prevent the cytokinin induced bud formation are fully functional in the moss P. patens (Knight et and the reversal of this effect by the calcium al, 1995). Thus, stress responses seem to be well ionophore A23l87 shows that the calcium rise is conserved between higher plants and mosses. essential for bud induction. The application of A23187 in fact leads to bud formation in the absence Auxin-transport and -binding Sites are Involved in of cytokinin (Saunders & Hepler, 1982). Inhibition of Auxin Response bud formation by antagonists of l,4-dihydropyridine Similar to the other auxin-induced responses, the (DHP) and the ability of DHP agonists to substitute bulk medium pH changes from an initial value of for the presence of cytokinin shows that DHP about 5 to 6.5 during IAA response. In the absence of sensitive voltage dependent calcium channels play an auxin, the caulonema can differentiate, but after a lag important role in the cytokinin response (Conrad & of 6 days, if the medium is buffered in the range of Hepler, 1988). G-proteins have been suggested to pH 5 - 5.5. The responsiveness of cells to auxin is also regulate these DHP sensitive calcium channels in modulated by medium pH and nutrient level (Johri & Physcomitrella (Schumaker & Gizinski, 1996). D'Souza, 1990). The lag is prolonged by the auxin There is a rise in intracellular calcium concen- antagonist p-chlorophenoxyisobutyric acid (PCIB) tration in Physcomitrella in response to physical which reduces polar, basipetal transport in Funaria stimuli such as cold shock and touch (Russell et al, rhizoids (Rose & Bopp, 1983) and is also known to 1996). Thus, similar calcium-sensing mechanisms compete with IAA for auxin-binding sites ( Jacobs & seem to exist in mosses and higher plants. As CDPKs Hertel, 1978). In the PCIB-treated cultures, there is no are the most predominant among the downstream inhibition of growth and in fact there is a profuse targets of calcium in plants, the regulation of calcium stimulation of secondary chloronema formation. Thus, regulated kinases from Funaria has emerged as the both basipetal transport and IAA-binding sites seem major thrust area. to be involved during caulonema differentiation and chloronema inhibition. Most recently, the initial Protein Kinasesfrom Chloronema Cells events in embryo development in Fucus distichus, a So far, at least five CDPKs and one calcium- brown alga, have also been found to be linked to regulated CaM dependent PK (CCaMK) have been auxin and auxin transport (Basu et al, 2002). characterised from chloronema cells. A calcium- JOHRI & D'SOUZA: HORMONAL REGULATION OF PROTONEMA DEVELOPMENT II independent 38 kDa MBP kinase regulated by ABA is independent of CaM and this response could be a part also present. The calcium is required for the of the calcium homeostasis mechanism. The autophosphorylation as well as substrate phosph- properties of the moss CCaMK are similar to lily and orylation by the CDPKs of Mr 44,48,63 and 70 kDa tobacco CCaMKs. The specific role of moss enzyme (D'Souza & Johri, 1999). There is an enhancement in is yet to be understood. the autophosphorylation of 44 kDa COPK in the presence of auxin or under other physiological Activation of a 38 kDa MBP Kinase by ABA conditions, which also lead to caulonema As mentioned earlier, a 38 kDa MBP kinase is differentiation in auxin-free medium. Thus, this PK activated in chloronema cells within minutes of ABA could have a role in caulonema formation. This PK is treatment (D'Souza & Johri, 2002a). The activation is recognised by moss anti-calmodulin antibodies and is transient, independent of calcium, specific to ABA also competed by the purified moss CaM during among phytohormones and is also evoked by salt immunoprecipitation showing that it has calmodulin- stress but not by other abiotic stresses. Salts and other like domain. compounds such as sugars, which change the The FhCDPK gene encodes a transcript of about intracellular water potential in the moss protonema, 2.6 kb which is upregulated by nutritional deprivation. also activate it (Unpublished data of 0' Souza & The genomic clone shows the canonical autoinhibitory Johri). The effects of ABA and NaCI are additive and region and the four EF hands (Mitra & Johri, 2000). both must be perceived independently and the signals The deduced amino acid sequence shows extensive converge at the level of MBP kinase. The moss MBP homology with other CDPKs namely, 73% identity kinase seems to be similar to the stress activated PKs with the Fragaria CDPK and 71 % homology with (SAPK) or other osmotically-activated PKs. The latter COPK isoform-7 of Arabidopsis. The homology to belong to the category of MAPKs such as the SIMK the liverwort Marchantia or the moss Tortula COPKs which is a salt-stress and pathogen induced MAPK was lower (59-64%). The codon usage in another recently described from Medicago sativa (Cardinale moss Physcomitrella is also similar to higher plants et al, 2002). Since ABA is also involved in conferring (Reski et al, 1998). Plants being monophyletic in resistance against anhydrobiosis in moss, the origin, the similarity of codon usage between Funaria overlapping activation of MBP kinase by salt and and Arabidopsis CDPK genes could reflect either ABA seems to be a part of signalling network. The convergent evolution since the acquisition of CDPK rapidity of the ABA response in Funaria indicates genes by these groups of plants or a lack of that as a survival strategy different from that of divergence of the codons. animals, the plant cells seem to be in a state of The 70 kOa moss CCaMK belongs to another very readiness capable of mounting a rapid initial response. important class of the calcium-regulated PKs that After the initial response, the cells seem to synthesize have a kinase domain followed by a CaM-binding new signaling proteins and other protective domain homologous to neural visinin-like molecules compounds. There is strong evidence for the ABA- (Harmon et al, 2000; Patil et al, 1995). The purified inducibility of the proteins sharing epitopes with the moss 70 kDa PK prefers lysine-rich histone as alpha subunits of the heterotrimeric G-proteins in substrate and is fully active in the presence of 50 ~M moss F. hygrometrica (Panigrahi, 1998). free calcium (D'Souza & Johri, 2002b). The enzyme is completely inactive at sub-optimal levels of free Manipulation of Calcium/CaM-dependent Protein calcium (23 ~M), but is activated by nanomolar levels Kinases and Phosphatases to Confer Stress of the moss CaM (5-100 nM). At higher CaM levels Tolerance in Plants (100-1000 nM, optimum 400 nM), the autophos- The ABA and abiotic stress signaling pathways are phorylation of the enzyme was also greatly stimulated networked in such a way that several key elements which in turn led to an enhanced substrate such as MAPKs, and CDPKs seem to be shared. phosphorylation. The activity of the moss CCaMK is Therefore, by manipulating the signal transduction thus CaM-dependent at low levels of calcium, a components or pathways, it should be possible to response which is likely to have a physiological confer or improve the stress tolerance in plants significance. At high calcium levels which are (Grover et al, 1999; Xing & Jordan, 2000). Monroy et nonphysiological, the moss enzyme becomes al (1993) demonstrated a role of calcium in the 12 INDIAN J BIOTECHNOL, JANUARY 2003

regulation of cold acclimation-specific genes in especially the role calciumlCaM dependent protein Medicago sativa. The COPKs have been found to be kinases and phosphatases, enables one to devise novel one of the positive regulators of tolerance to salt and and viable strategies for engineering tolerance against cold stresses in several plants. Maize mesophyll stresses. As the protein kinases such as COPKs protoplasts expressing the GFP reporter gene driven operate early in the signaling pathway, their by an ABA-inducible HVAI promoter from barley manipulation also makes it possible to regulate the show an enhanced expression in response to ABA, activity of several genes that function downstream. salt and cold stresses and darkness. On co-transfecting these protoplasts with constitutively active forms of Origin of Hormonal Response in Plants the Arabidopsis COPKI and COPKla, the GFP Distribution of Phytohormones and their Main Role expression was found even in the absence of stress The major groups of phytohormones are more or signals or ABA. Thus, activated COPKs bypass the less ubiquitously distributed in plants. Auxin, requirement of the signals in evoking the stress cytokinin, ethylene, gibberellins and ABA have been signaling (Sheen, 1996). The over-expression of rice reported from algae (Jacobs, 1986; Johri, 1990), but cold- and salt-inducible OsCOPK7, conferred the evidence for their hormonal role is not tolerance against both the stresses in transgenic rice unequivocal. It is only in the bryophytes that besides (Saijo et al, 2000). The COPKs have also been found the presence, specific responses to above hormones to be involved in the defense response against the have been demonstrated. The evidence for the fungus Cladosporium fulvum in resistant tobacco presence and for specific effects of gibberellins in (Romeis et al, 2000); mechanical strain and bryophytes is however, not very strong. The dehydration stress in mung bean and Arabidopsis regulatory role of gibberellins as antheridiogens is respectively (Botella et al, 1996; Urao et al, 1994). well documented in some of the of the family Among other protein kinases, an Arabidopsis Schizaeaceae (Bopp, 1990; Johri, 1990). homologue of GSK3/shaggy-like kinase, AtGSK1, is involved in salt stress responses (Piao et al, 2001). The basic responses of auxin, cytokinin, ABA and The transgenic plants over-expressing AtGSKI ethylene are remarkably conserved in plants. The role showed enhanced tolerance to salinity and the NaCI of auxin in caulonema differentiation has already been responsive genes were induced in the absence of salt. mentioned. Caulonema is similar to the rhizoids and There is also evidence for the involvement of both represent the same cell type, which is the other proteins such as calciumlCaM-dependent forerunner of root system of tracheophytes (vascular phosphatases--the calcineurin, in the stress tolerance. plants). Likewise, the cytokinin-induced bud initial The expression of AtCBLl (Arabidopsis thaliana cell, which develops into a three-sided apical cell calcineurin B-like protein) is induced by stresses such forming the moss gametophore, is analogous to the as cold, drought and wounding (Kudla et al, 1999). initial cell or a founder cell of a multicellular apical Another gene with considerable homology to meristem. Thus, auxin and cytokinin evoke calcineurin B, is the salt overly sensitive 3 (SOS3) respectively the rhizogenic and shoot bud-forming gene of Arabidopsis. It has been found to mediate responses in plants. Similar to the higher plants, ABA calcium signaling associated with the acquisition of also arrests development and confers tolerance against cold tolerance (Liu and Zhu, 1997). SOS3 interacts water stress in mosses and at the biochemical level at with a serine threonine kinase, the SOS2 and upon least the action mechanism of ABA action seems' to increase in cytosolic calcium in response to high salt be highly conserved. Ethylene is produced by axenic stress, the activated SOS2/S0S3 complex seems to cultures of several species of algae, liverworts, modify the Na+ and K+ transporters thereby causing mosses and ferns and in general retards cell division salt tolerance (Halfter et al, 2000). Likewise, the but promotes cell elongation (Johri, 1990). It transgenic tobacco plants coexpressing the catalytic suppresses the ventral row of leaves in the leafy and regulatory subunits of yeast calcineurin showed liverwort, Plagiochila arctica (Basile & Basile, an enhanced tolerance to salt stress (Pardo et al, 1983). The elongation of seta in the sporophyte of the 1998). thallose liverwort Pellia epiphylla involves a dual regulation by auxin and ethylene. The elongating The above account shows that a deeper setae contain adequate endogenous auxin and at the understanding of the signaling mechanisms and same time can also respond to exogenous auxin. JOHRl & D'SOUZA: HORMONAL REGULATION OF PROTONEMA DEVELOPMENT 13

Elongating setae release more ethylene than those not (Schraudolf, 1985). He further argues that "In contrast undergoing elongation (Thomas et al, 1983), a feature to animal evolution, the phylogeny of plants seems to characteristic of auxin-rich tissues undergoing cell be characterized by a post-evolution of hormone- elongation. Auxin application also enhances ethylene receptor molecule. This event was a prerequisite for a production in P. arctica (Law et al, 1985). In the common and ubiquitously distributed metabolic moss F. hygrometrica, the formation of tnema cells in product to take over the function of a regulatory old cultures seems to be related to ethylene molecule. The phylogeny of phytohormones therefore production (Rohwer & Bopp, 1985). Ethylene has has to be written as a phylogeny of their receptors" also been found to promote megasporangium (Schraudolf, 1985). Following the same argument, we formation in the lycopod Selaginella wallacei wish to propose that it is not difficult to visualise the presumably by blocking the final cell division of the origin of specific receptors or the high-affinity sporogenous tissue cells (Brooks, 1973). Similar to its perception mechanisms at the level of the ancestor of effects in some of the aquatic higher plants, ethylene land plants and the consequent acquisition of application stimulates the elongation of frond rachis hormonal role by auxin, cytokinin, ABA, ethylene in the semi-aquatic Regnellidium diphyllum and gibberellins. In Arabidopsis the receptors for (Walters & Osborne, 1979). There is thus ethylene and cytokinin have been identified to be the overwhelming evidence for the remarkably conserved sensory hybrid-type histidine kinase and the two- ethylene effects between cryptogams and angiosperms component systems are involved in the signal and an interaction with auxin seems to be involved in transduction (Hwang & Sheen, 2001; Urao et al, many cases. 2000). The ethylene receptor acts upstream of a Raf protein kinase (a mitogen activated protein kinase Possible Acquisition of Hormonal Function by kinase kinase or MAPKKK), and the overall signaling Secondary Metabolites pathway is similar to the osmoregulation pathway in Growth substances acting as phytohormones are yeast. The two-component histidine receptors seems also present in several bacteria and fungi as products to have been derived from the cyanobacterial genome of secondary metabolism and the notion of a as a result of horizontal transfer of genes during the metabolite acquiring a signaling or a hormonal origin of the ancestor of plants (Meyerowitz, 2002; function has received some attention. Some of the sex Urao et al, 2000). The receptors for the other hormones (sexual pheromones) of algae and fungi hormones have yet to be identified. As bryophytes have been characterised chemically (Kochert, 1978; represent the earliest group of land plants with AI-Hasani & Jaenicke, 1992) and chemical signaling hormonal responses, we expect the histidine protein through pheromones had already evolved in the kinase receptors to be present in them and clones sexual reproduction and somatic cell repair in algae homologous with ethylene receptor have been (Waaland, 1986). According to Kochert (1978), the reported from P. patens (Fujiwara & Tohe, 2001). pheromones of unicellular eucarionts could be the The auxin transport and biosynthesis mechanisms in ancestors of hormones for all multicellular eucarionts. the protonema of Funaria are comparable to higher The action of secondary metabolites (present in plants and are thus highly conserved (Rose & Bopp, ancestral forms) as hormones could have acquired a 1983; Jayaswal & Johri, 1985). regulatory role with the evolution of multicellular orgamsms. Many Signaling Components could have Evolved at the Level of Common Ancestor Origin of Specific Receptors All land plants, the embryophytes, are believed to Following Kochert's general idea about the have a monophyletic origin. Based on the possible origin of hormones from pheromones, comparative morphology and molecular phylogeny, Schraudolf (1985, 1986) has compared the similarities the liverworts are believed to be basal and distinct. between the pheromone system of Schizaeaceae and and either mosses or hornworts represent a living the gibberellin responses in seed plants. According to sister group to vascular plants (Kendric & Crane him, "the reaction of antheridiogens in Schizaeaceous 1997). Among the most recent studies, the analysis of ferns represents the 'moment of becoming a hormone' the mitochondrial nadl gene by Hashimoto & Sato for gibberellin like molecules in phanerogams" (2001) suggests the monophyly of mosses and 14 INDIAN J BIOTECHNOL, JANUARY 2003 tracheophytes and the paraphyly of liverworts to these Conclusions two taxa. It has been proposed that during the The moss protonema has emerged as an excellent transition from an aqueous to the gaseous medium, developmental system to study the signaling the important metabolic pathways including the plant mechanisms during hormonal action and responses to hormones in tracheophytes (vascular plants) arose abiotic stresses. The overall hormonal regulation of from the pre-existing elements of primary metabolism cell differentiation involves several interacting in charophycean algae and bryophytes. Due to lack of factors--both inhibitory and the promo tory ones. The information, it is difficult to comment about the side branch initial on a caulonema filament is presence of pre-existing elements of hormonal pluripotent and depending on the phytohormone mechanisms in charophycean algae, but as already applied it can differentiate either into a chloronemal, a discussed, there is now unequivocal evidence for the caulonemal or a bud initial cell. The role of a origin of ethylene and cytokinin receptors from particular hormone can thus be visualized in histidine kinase of blue green algae. As already channeling or fixing the terminal destiny of a progeny mentioned, the auxin transport and biosynthetic cell to a specific developmental fate by inhibiting the pathways seem to be conserved in moss and higher others. The available information suggests that the plants; it is plausible that the similarities extend specific responses to hormones such as auxin, beyond this to the level of signaling components and cytokinin and ABA could have evolved early at the transduction mechanisms involving calcium! level of bryophytes and even in these cryptogams the calmodulin, CDPKs and MAP kinases. Interestingly, level of overall mechanism of their action seems the presence of similar hormonal mechanisms in the comparable to that of the higher plants. Based on mosses and seed plants, the two plant groups that had similar basipetal polar transport and auxin effects evolved and diverged half a billion years ago, Hertel (1983), had visualised the importance of suggests that the modules were possibly present in the mosses to understand the origin, differentiation and presumed unicellular common ancestor of plants. As diversification of transport and action of plant growth similar signaling mechanisms are utilized in the substances. The stage seems to be set towards a fuller animals also, the signaling elements or components of realisation of this idea. The partial sequence of signal transduction must have evolved and been Funaria calcium-dependent PK gene shows extensive present before the separation of plant and animal homology with CDPK genes isolated from higher lineages. For instance the calcium signaling including plants. Reski et al (1998) had also observed a high CaM operates both in plants and animals. The CDPKs degree of conservation between moss and seed plant on the other hand, are specific to plants and must have sequences and the codon usage in moss P. patens is evolved later in the ancestor of plants only. Besides very similar to that of the dicotyledenous plants. We plants, sequences coding for LEA proteins are also need to learn more about the identity of other present in the genomes of micro-organisms and signaling molecules in plants. The analysis of protein nematode and could have originated in the ancient kinase genes in the genome of mosses is likely to cell types as a common strategy against anhydrobiosis provide additional and new information about the (Browne et al, 2002). The available evidence thus origin and diversification of histidine kinase type of suggests that the receptors and signaling elements in receptors. With the demonstration of highly efficient the extant plants had their origin from the elements in homologous recombination in P. patens (Schaefer & the microbial and other ancient organisms. These Zryd, 1997), a far more rapid progress can be elements are now regulated by the eukaryotic expected in future. promoters and have been shuffled around to generate different cascades. As pointed out by Schraudolf (1985), the crux of the issue in phytohormonal References responses is the problem of the origin of specific Ainapure S 0, 1998. Studies on the Abscisic acid and stress- receptors. With rapid advances in molecular induced proteins in the protonema of the moss Funaria hygrometrica. MSc Thesis. University of Mumbai. Mumbai. phylogeny hormonal signaling pathways, it may Pp 1-57. become possible to trace the events that ultimately Al-Hasani H & Jaenicke L, 1992. 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