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

Hormonal Regulation of Moss Protonema Development and the Possible Origin ofPlant Hormonal Responses in Bryophytes

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

The protonema of mosses is a far simpler paradigm to understand the mechanism of hormonal action andtolerance to abiotic stresses in plants. Its developmental biology, responses to hormones and the similarity ofsignaling mechanisms with higher plants are reviewed. There is strong evidence for second messenger role of calciumions 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, thefour EF hands and the highest homology to CDPKs from higher plants but far lower to liverwort or other mossCDPK 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 promoteris fully functional in mosses, the ABA signaling mechanism seems to be highly conserved. In plants, the CDPKs areinvolved not only in hormonal signaling but also in the acclimation response against abiotic stresses. Themanipulation of signal transduction components such as transcription factors, CDPKs and calcineurin have emergedas viable strategies to genetically engineer the stress tolerant plants. There is increasing evidence to support theorigin of plant hormonal responses at the level of bryophytes.

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

IntroductionThe overall development in plants is regulated by

environmental and internal factors. How plantsperceive various signals, transduce them andultimately alter the growth in terms of temporal .andspatial patterns, is beginning to be comprehended.The bryophytes represent the earliest group of landplants where the phytohormone-mediated morpho-genetic responses comparable to that in higher plantshave been found (Bopp, 1990; Johri, 1990). Theprotonema of mosses such as Funaria hygrometricaHedw. and Physcomitrella patens (Hedw.) B.S.G.have been used to study the hormonal regulation ofdevelopment (Bopp & Atzorn, 1992; Cove & Knight,

*Author for correspondence:Tel: 022-22152971 Ext-2255 ; Fax: 022-22152110E-mail: mmjohri@mailhosuifr.res.inAbbreviations:A23187: calcium ionophore; ABA: abscisic acid; CaM: calmodulin;CCaMK: calcium regulated calmodulin-dependent PK; CDPK:calcium-dependent protein kinase; CTC: chlorotetracycline; DHP:IA-dihydropyridine; FhCDPK: Funaria hygrometrica CDPK gene;LEA: late embryogenesis abundant; MAPKKK: mitogen activatedprotein kinase kinase kinase; MBP kinase: myelin basic proteinkinase; PCIB: p-chlorophenoxyisobutyric acid; PK: Protein kinase;SAPK: stress activated PK; SIMK: salt stress-induced MAPK.

1993; Johri, 1978). The hormonal responses are rapidand discernible either in single cells or in a group of afew cells and are thus more or less cell autonomous.

This review focusses on the hormonal regulation ofprotonema development, the diversity of proteinkinases (PKs) from the moss, F. hygrometrica and theimportance of PKs in relation to abiotic stresstolerance in plants. The possible origin of hormonalresponses is also discussed.

Hormonal Regulation of Cell differentiation in MossSuspension Cultures

A remarkable feature of the protonemal suspensioncultures of Funaria has been the long-term stability ofthe cell line J-2 which has now been maintained forover 32 years by repeated subculture in a low-calciummedium. During this long time, it has neither lost thepotential to differentiate nor the responsiveness tophytohormones (Johri, 1974). The caulonema differen-tiation is regulated by inoculum size, auxin concen-tration and nutrient level. It marks a major develop-mental switch, which is turned on by biologicallyactive auxins and ethyl ester of IAA but not byindoleacrylic acid or 2,4-dichlorophenoxyacetic acid(Johri & Desai, 1973; Johri & D'Souza, 1990). There is

10 INDIAN J BIOTECHNOL, JANUARY 2003

an increase In endogenous auxin during caulonemaformation (Atzorn et al, 1989a, 1989b). Thecytokinins induce the formation of bud initials, whicharise as side branches from caulonemal cells (Gorton& Eakin, 1957). Cytokinin-over-producing mutants ofP. patens form the buds constitutively (Ashton et al,1979).

ABA inhibits the growth and cytokinin-inducedbud formation (Valdon & Mummery, 1971). ABA-treatment of protonemal cells is reported to increasetolerance against freezing and to confer adaptation todrought (Nagao et al, 2001; Werner et al, 1993). Theendogenous level of ABA increases in moss plantsunder arid conditions (Bopp & Werner, 1993) and anapplication of ABA causes closure of stomata in theFunaria sporophyte (Garner & Paolillo, 1973). In thepresence of ABA, the side branches on the protonemaremain short and develop into resting structuresreferred to as the brood cells (Johri, 1988). Formationof new polypeptides which share epitopes with thehigher plant LEA (late embryogenesis abundant)proteins, have been demonstrated in the protonema(Ainapure, 1998). The ABA- and osmotic stress-inducible promoter elements from the wheat Em geneare fully functional in the moss P. patens (Knight etal, 1995). Thus, stress responses seem to be wellconserved between higher plants and mosses.

Auxin-transport and -binding Sites are Involved inAuxin Response

Similar to the other auxin-induced responses, thebulk medium pH changes from an initial value ofabout 5 to 6.5 during IAA response. In the absence ofauxin, the caulonema can differentiate, but after a lagof 6 days, if the medium is buffered in the range ofpH 5 - 5.5. The responsiveness of cells to auxin is alsomodulated by medium pH and nutrient level (Johri &D'Souza, 1990). The lag is prolonged by the auxinantagonist p-chlorophenoxyisobutyric acid (PCIB)which reduces polar, basipetal transport in Funariarhizoids (Rose & Bopp, 1983) and is also known tocompete with IAA for auxin-binding sites ( Jacobs &Hertel, 1978). In the PCIB-treated cultures, there is noinhibition of growth and in fact there is a profusestimulation of secondary chloronema formation. Thus,both basipetal transport and IAA-binding sites seemto be involved during caulonema differentiation andchloronema inhibition. Most recently, the initialevents in embryo development in Fucus distichus, abrown alga, have also been found to be linked toauxin and auxin transport (Basu et al, 2002).

The Role of Calcium in MossesThe role of calcium in cytokinin induced bud

formation in Funaria and Physcomitrella is well-documented (Schumaker & Dietrich, 1997). Using thelipophilic fluorescent calcium chelating probe--chlorotetracycline (CTC), a calcium rise followingcytokinin treatment was localised to the presumptiveinitial cell site (Saunders & Hepler, 1981).Measurements of calcium currents using vibratingmicroelectrodes along a caulonema filament haveshown that cytokinin application leads to an increasein the magnitude of the inward current and causes achange in the spatial properties of the current(Saunders, 1986). In a caulonemal cell maximuminward current is observed near the nucleus but aftercytokinin application, there is a decrease in thecurrent near the centre which is followed by a rise inthe apical end. This rise in the inward current predictsthe location of the initial cell and falls to resting levelswith the onset of the initial cell outgrowth. Thiscurrent can be blocked by gadolinium, whichcompetitively inhibits calcium transport.

The calcium channel blockers, verapamil andD-600 prevent the cytokinin induced bud formationand the reversal of this effect by the calciumionophore A23l87 shows that the calcium rise isessential for bud induction. The application ofA23187 in fact leads to bud formation in the absenceof cytokinin (Saunders & Hepler, 1982). Inhibition ofbud formation by antagonists of l,4-dihydropyridine(DHP) and the ability of DHP agonists to substitutefor the presence of cytokinin shows that DHPsensitive voltage dependent calcium channels play animportant role in the cytokinin response (Conrad &Hepler, 1988). G-proteins have been suggested toregulate these DHP sensitive calcium channels inPhyscomitrella (Schumaker & Gizinski, 1996).

There is a rise in intracellular calcium concen-tration in Physcomitrella in response to physicalstimuli such as cold shock and touch (Russell et al,1996). Thus, similar calcium-sensing mechanismsseem to exist in mosses and higher plants. As CDPKsare the most predominant among the downstreamtargets of calcium in plants, the regulation of calciumregulated kinases from Funaria has emerged as themajor thrust area.

Protein Kinases from Chloronema CellsSo far, at least five CDPKs and one calcium-

regulated CaM dependent PK (CCaMK) have beencharacterised from chloronema cells. A calcium-

JOHRI & D'SOUZA: HORMONAL REGULATION OF PROTONEMA DEVELOPMENT

independent 38 kDa MBP kinase regulated by ABA isalso present. The calcium is required for theautophosphorylation as well as substrate phosph-orylation by the CDPKs of Mr 44,48,63 and 70 kDa(D'Souza & Johri, 1999). There is an enhancement inthe autophosphorylation of 44 kDa COPK in thepresence of auxin or under other physiologicalconditions, which also lead to caulonemadifferentiation in auxin-free medium. Thus, this PKcould have a role in caulonema formation. This PK isrecognised by moss anti-calmodulin antibodies and isalso competed by the purified moss CaM duringimmunoprecipitation showing that it has calmodulin-like domain.

The FhCDPK gene encodes a transcript of about2.6 kb which is upregulated by nutritional deprivation.The genomic clone shows the canonical autoinhibitoryregion and the four EF hands (Mitra & Johri, 2000).The deduced amino acid sequence shows extensivehomology with other CDPKs namely, 73% identitywith the Fragaria CDPK and 71 % homology withCOPK isoform-7 of Arabidopsis. The homology tothe liverwort Marchantia or the moss Tortula COPKswas lower (59-64%). The codon usage in anothermoss Physcomitrella is also similar to higher plants(Reski et al, 1998). Plants being monophyletic inorigin, the similarity of codon usage between Funariaand Arabidopsis CDPK genes could reflect eitherconvergent evolution since the acquisition of CDPKgenes by these groups of plants or a lack ofdivergence of the codons.

The 70 kOa moss CCaMK belongs to another veryimportant class of the calcium-regulated PKs thathave a kinase domain followed by a CaM-bindingdomain homologous to neural visinin-like molecules(Harmon et al, 2000; Patil et al, 1995). The purifiedmoss 70 kDa PK prefers lysine-rich histone assubstrate and is fully active in the presence of 50 ~Mfree calcium (D'Souza & Johri, 2002b). The enzymeis completely inactive at sub-optimal levels of freecalcium (23 ~M), but is activated by nanomolar levelsof the moss CaM (5-100 nM). At higher CaM levels(100-1000 nM, optimum 400 nM), the autophos-phorylation of the enzyme was also greatly stimulatedwhich in turn led to an enhanced substratephosphorylation. The activity of the moss CCaMK isthus CaM-dependent at low levels of calcium, aresponse which is likely to have a physiologicalsignificance. At high calcium levels which arenonphysiological, the moss enzyme becomes

II

independent of CaM and this response could be a partof the calcium homeostasis mechanism. Theproperties of the moss CCaMK are similar to lily andtobacco CCaMKs. The specific role of moss enzymeis yet to be understood.

Activation of a 38 kDa MBP Kinase by ABAAs mentioned earlier, a 38 kDa MBP kinase is

activated in chloronema cells within minutes of ABAtreatment (D'Souza & Johri, 2002a). The activation istransient, independent of calcium, specific to ABAamong phytohormones and is also evoked by saltstress but not by other abiotic stresses. Salts and othercompounds such as sugars, which change theintracellular water potential in the moss protonema,also activate it (Unpublished data of 0'Souza &Johri). The effects of ABA and NaCI are additive andboth must be perceived independently and the signalsconverge at the level of MBP kinase. The moss MBPkinase seems to be similar to the stress activated PKs(SAPK) or other osmotically-activated PKs. The latterbelong to the category of MAPKs such as the SIMKwhich is a salt-stress and pathogen induced MAPKrecently described from Medicago sativa (Cardinaleet al, 2002). Since ABA is also involved in conferringresistance against anhydrobiosis in moss, theoverlapping activation of MBP kinase by salt andABA seems to be a part of signalling network. Therapidity of the ABA response in Funaria indicatesthat as a survival strategy different from that ofanimals, the plant cells seem to be in a state ofreadiness capable of mounting a rapid initial response.After the initial response, the cells seem to synthesizenew signaling proteins and other protectivecompounds. There is strong evidence for the ABA-inducibility of the proteins sharing epitopes with thealpha subunits of the heterotrimeric G-proteins inmoss F. hygrometrica (Panigrahi, 1998).

Manipulation of Calcium/CaM-dependent ProteinKinases and Phosphatases to Confer StressTolerance in Plants

The ABA and abiotic stress signaling pathways arenetworked in such a way that several key elementssuch as MAPKs, and CDPKs seem to be shared.Therefore, by manipulating the signal transductioncomponents or pathways, it should be possible toconfer or improve the stress tolerance in plants(Grover et al, 1999; Xing & Jordan, 2000). Monroy etal (1993) demonstrated a role of calcium in the

12 INDIAN J BIOTECHNOL, JANUARY 2003

regulation of cold acclimation-specific genes inMedicago sativa. The COPKs have been found to beone of the positive regulators of tolerance to salt andcold stresses in several plants. Maize mesophyllprotoplasts expressing the GFP reporter gene drivenby an ABA-inducible HVAI promoter from barleyshow an enhanced expression in response to ABA,salt and cold stresses and darkness. On co-transfectingthese protoplasts with constitutively active forms ofthe Arabidopsis COPKI and COPKla, the GFPexpression was found even in the absence of stresssignals or ABA. Thus, activated COPKs bypass therequirement of the signals in evoking the stresssignaling (Sheen, 1996). The over-expression of ricecold- and salt-inducible OsCOPK7, conferredtolerance against both the stresses in transgenic rice(Saijo et al, 2000). The COPKs have also been foundto be involved in the defense response against thefungus Cladosporium fulvum in resistant tobacco(Romeis et al, 2000); mechanical strain anddehydration stress in mung bean and Arabidopsisrespectively (Botella et al, 1996; Urao et al, 1994).Among other protein kinases, an Arabidopsishomologue of GSK3/shaggy-like kinase, AtGSK1, isinvolved in salt stress responses (Piao et al, 2001).The transgenic plants over-expressing AtGSKIshowed enhanced tolerance to salinity and the NaCIresponsive genes were induced in the absence of salt.There is also evidence for the involvement ofother proteins such as calciumlCaM-dependentphosphatases--the calcineurin, in the stress tolerance.The expression of AtCBLl (Arabidopsis thalianacalcineurin B-like protein) is induced by stresses suchas cold, drought and wounding (Kudla et al, 1999).Another gene with considerable homology tocalcineurin B, is the salt overly sensitive 3 (SOS3)gene of Arabidopsis. It has been found to mediatecalcium signaling associated with the acquisition ofcold tolerance (Liu and Zhu, 1997). SOS3 interactswith a serine threonine kinase, the SOS2 and uponincrease in cytosolic calcium in response to high saltstress, the activated SOS2/S0S3 complex seems tomodify the Na+ and K+ transporters thereby causingsalt tolerance (Halfter et al, 2000). Likewise, thetransgenic tobacco plants coexpressing the catalyticand regulatory subunits of yeast calcineurin showedan enhanced tolerance to salt stress (Pardo et al,1998).

The above account shows that a deeperunderstanding of the signaling mechanisms and

especially the role calciumlCaM dependent proteinkinases and phosphatases, enables one to devise noveland viable strategies for engineering tolerance againststresses. As the protein kinases such as COPKsoperate early in the signaling pathway, theirmanipulation also makes it possible to regulate theactivity of several genes that function downstream.

Origin of Hormonal Response in PlantsDistribution of Phytohormones and their Main Role

The major groups of phytohormones are more orless ubiquitously distributed in plants. Auxin,cytokinin, ethylene, gibberellins and ABA have beenreported from algae (Jacobs, 1986; Johri, 1990), butthe evidence for their hormonal role is notunequivocal. It is only in the bryophytes that besidesthe presence, specific responses to above hormoneshave been demonstrated. The evidence for thepresence and for specific effects of gibberellins inbryophytes is however, not very strong. Theregulatory role of gibberellins as antheridiogens iswell documented in some of the ferns of the familySchizaeaceae (Bopp, 1990; Johri, 1990).

The basic responses of auxin, cytokinin, ABA andethylene are remarkably conserved in plants. The roleof auxin in caulonema differentiation has already beenmentioned. Caulonema is similar to the rhizoids andboth represent the same cell type, which is theforerunner of root system of tracheophytes (vascularplants). Likewise, the cytokinin-induced bud initialcell, which develops into a three-sided apical cellforming the moss gametophore, is analogous to theinitial cell or a founder cell of a multicellular apicalmeristem. Thus, auxin and cytokinin evokerespectively the rhizogenic and shoot bud-formingresponses in plants. Similar to the higher plants, ABAalso arrests development and confers tolerance againstwater stress in mosses and at the biochemical level atleast the action mechanism of ABA action seems' tobe highly conserved. Ethylene is produced by axeniccultures of several species of algae, liverworts,mosses and ferns and in general retards cell divisionbut promotes cell elongation (Johri, 1990). Itsuppresses the ventral row of leaves in the leafyliverwort, Plagiochila arctica (Basile & Basile,1983). The elongation of seta in the sporophyte of thethallose liverwort Pellia epiphylla involves a dualregulation by auxin and ethylene. The elongatingsetae contain adequate endogenous auxin and at thesame time can also respond to exogenous auxin.

JOHRl & D'SOUZA: HORMONAL REGULATION OF PROTONEMA DEVELOPMENT

Elongating setae release more ethylene than those notundergoing elongation (Thomas et al, 1983), a featurecharacteristic of auxin-rich tissues undergoing cellelongation. Auxin application also enhances ethyleneproduction in P. arctica (Law et al, 1985). In themoss F. hygrometrica, the formation of tnema cells inold cultures seems to be related to ethyleneproduction (Rohwer & Bopp, 1985). Ethylene hasalso been found to promote megasporangiumformation in the lycopod Selaginella wallaceipresumably by blocking the final cell division of thesporogenous tissue cells (Brooks, 1973). Similar to itseffects in some of the aquatic higher plants, ethyleneapplication stimulates the elongation of frond rachisin the semi-aquatic fern Regnellidium diphyllum(Walters & Osborne, 1979). There is thusoverwhelming evidence for the remarkably conservedethylene effects between cryptogams and angiospermsand an interaction with auxin seems to be involved inmany cases.

Possible Acquisition of Hormonal Function bySecondary Metabolites

Growth substances acting as phytohormones arealso present in several bacteria and fungi as productsof secondary metabolism and the notion of ametabolite acquiring a signaling or a hormonalfunction has received some attention. Some of the sexhormones (sexual pheromones) of algae and fungihave been characterised chemically (Kochert, 1978;AI-Hasani & Jaenicke, 1992) and chemical signalingthrough pheromones had already evolved in thesexual reproduction and somatic cell repair in algae(Waaland, 1986). According to Kochert (1978), thepheromones of unicellular eucarionts could be theancestors of hormones for all multicellular eucarionts.The action of secondary metabolites (present inancestral forms) as hormones could have acquired aregulatory role with the evolution of multicellularorgamsms.

Origin of Specific ReceptorsFollowing Kochert's general idea about the

possible origin of hormones from pheromones,Schraudolf (1985, 1986) has compared the similaritiesbetween the pheromone system of Schizaeaceae andthe gibberellin responses in seed plants. According tohim, "the reaction of antheridiogens in Schizaeaceousferns represents the 'moment of becoming a hormone'for gibberellin like molecules in phanerogams"

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(Schraudolf, 1985). He further argues that "In contrastto animal evolution, the phylogeny of plants seems tobe characterized by a post-evolution of hormone-receptor molecule. This event was a prerequisite for acommon and ubiquitously distributed metabolicproduct to take over the function of a regulatorymolecule. The phylogeny of phytohormones thereforehas to be written as a phylogeny of their receptors"(Schraudolf, 1985). Following the same argument, wewish to propose that it is not difficult to visualise theorigin of specific receptors or the high-affinityperception mechanisms at the level of the ancestor ofland plants and the consequent acquisition ofhormonal role by auxin, cytokinin, ABA, ethyleneand gibberellins. In Arabidopsis the receptors forethylene and cytokinin have been identified to be thesensory hybrid-type histidine kinase and the two-component systems are involved in the signaltransduction (Hwang & Sheen, 2001; Urao et al,2000). The ethylene receptor acts upstream of a Rafprotein kinase (a mitogen activated protein kinasekinase kinase or MAPKKK), and the overall signalingpathway is similar to the osmoregulation pathway inyeast. The two-component histidine receptors seemsto have been derived from the cyanobacterial genomeas a result of horizontal transfer of genes during theorigin of the ancestor of plants (Meyerowitz, 2002;Urao et al, 2000). The receptors for the otherhormones have yet to be identified. As bryophytesrepresent the earliest group of land plants withhormonal responses, we expect the histidine proteinkinase receptors to be present in them and cloneshomologous with ethylene receptor have beenreported from P. patens (Fujiwara & Tohe, 2001).The auxin transport and biosynthesis mechanisms inthe protonema of Funaria are comparable to higherplants and are thus highly conserved (Rose & Bopp,1983; Jayaswal & Johri, 1985).

Many Signaling Components could have Evolved atthe Level of Common Ancestor

All land plants, the embryophytes, are believed tohave a monophyletic origin. Based on thecomparative morphology and molecular phylogeny,the liverworts are believed to be basal and distinct.and either mosses or hornworts represent a livingsister group to vascular plants (Kendric & Crane1997). Among the most recent studies, the analysis ofthe mitochondrial nadl gene by Hashimoto & Sato(2001) suggests the monophyly of mosses and

14 INDIAN J BIOTECHNOL, JANUARY 2003

tracheophytes and the paraphyly of liverworts to thesetwo taxa. It has been proposed that during thetransition from an aqueous to the gaseous medium,the important metabolic pathways including the planthormones in tracheophytes (vascular plants) arosefrom the pre-existing elements of primary metabolismin charophycean algae and bryophytes. Due to lack ofinformation, it is difficult to comment about thepresence of pre-existing elements of hormonalmechanisms in charophycean algae, but as alreadydiscussed, there is now unequivocal evidence for theorigin of ethylene and cytokinin receptors fromhistidine kinase of blue green algae. As alreadymentioned, the auxin transport and biosyntheticpathways seem to be conserved in moss and higherplants; it is plausible that the similarities extendbeyond this to the level of signaling components andtransduction mechanisms involving calcium!calmodulin, CDPKs and MAP kinases. Interestingly,the presence of similar hormonal mechanisms in themosses and seed plants, the two plant groups that hadevolved and diverged half a billion years ago,suggests that the modules were possibly present in thepresumed unicellular common ancestor of plants. Assimilar signaling mechanisms are utilized in theanimals also, the signaling elements or components ofsignal transduction must have evolved and beenpresent before the separation of plant and animallineages. For instance the calcium signaling includingCaM operates both in plants and animals. The CDPKson the other hand, are specific to plants and must haveevolved later in the ancestor of plants only. Besidesplants, sequences coding for LEA proteins are alsopresent in the genomes of micro-organisms andnematode and could have originated in the ancientcell types as a common strategy against anhydrobiosis(Browne et al, 2002). The available evidence thussuggests that the receptors and signaling elements inthe extant plants had their origin from the elements inthe microbial and other ancient organisms. Theseelements are now regulated by the eukaryoticpromoters and have been shuffled around to generatedifferent cascades. As pointed out by Schraudolf(1985), the crux of the issue in phytohormonalresponses is the problem of the origin of specificreceptors. With rapid advances in molecularphylogeny hormonal signaling pathways, it maybecome possible to trace the events that ultimatelyculminated in the origin of hormonal perception andsignal transduction mechanisms in plants.

Conclusions

The moss protonema has emerged as an excellentdevelopmental system to study the signalingmechanisms during hormonal action and responses toabiotic stresses. The overall hormonal regulation ofcell differentiation involves several interactingfactors--both inhibitory and the promo tory ones. Theside branch initial on a caulonema filament ispluripotent and depending on the phytohormoneapplied it can differentiate either into a chloronemal, acaulonemal or a bud initial cell. The role of aparticular hormone can thus be visualized inchanneling or fixing the terminal destiny of a progenycell to a specific developmental fate by inhibiting theothers. The available information suggests that thespecific responses to hormones such as auxin,cytokinin and ABA could have evolved early at thelevel of bryophytes and even in these cryptogams thelevel of overall mechanism of their action seemscomparable to that of the higher plants. Based onsimilar basipetal polar transport and auxin effectsHertel (1983), had visualised the importance ofmosses to understand the origin, differentiation anddiversification of transport and action of plant growthsubstances. The stage seems to be set towards a fullerrealisation of this idea. The partial sequence ofFunaria calcium-dependent PK gene shows extensivehomology with CDPK genes isolated from higherplants. Reski et al (1998) had also observed a highdegree of conservation between moss and seed plantsequences and the codon usage in moss P. patens isvery similar to that of the dicotyledenous plants. Weneed to learn more about the identity of othersignaling molecules in plants. The analysis of proteinkinase genes in the genome of mosses is likely toprovide additional and new information about theorigin and diversification of histidine kinase type ofreceptors. With the demonstration of highly efficienthomologous recombination in P. patens (Schaefer &Zryd, 1997), a far more rapid progress can beexpected in future.

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