PlantCell Reports

Plant Cell Reports (1996) 15:495-499

9 Springer-Verlag 1996

Polyamine biosynthesis during somatic embryogenesis in interior spruce (Picea glauca x Picea engelmannii complex) Vindhya Amarasinghe, Rajwinder Dhami, and John E. Carlson Biotechnology Laboratory, # 237-6174 University Boulevard, University of British Columbia, Vancouver B.C., Canada V6T IZ3 Received 25 March 1995/Revised version received 18 August 1995 - Communicated by F. Congtabel

Abstract Putrescine, spermidine, and spennine levels during somatic embryogenesis of interior spruce (Picea glauca x Picea engelmannii complex) were quantified. On abscisic acid supplemented growth medium pu~xescineand spennidine levels increased two-fold coinciding with maturation of the early somatic embryos to globular embryos. Polyclonal antibodies raised against Eschefichia coil arginine decarboxylase (ADC) and omithine decarboxylase (ODC), following affimty purification specifically recognized spruce ADC and ODC, which corresponded to 85kD and 65kD bands on western blots of total protein extracts from embryogenic masses. Immunoassays using these anl~txxlies showed increased ADC levels corresponding to embryo maturation while ODC levels remained the same. From these results it is concluded that polyamines are involved in the maturation of somatic embryos of interior spruce. Abbreviations: ADC, arginine decarboxylase; BSA, bovine serum albumin; ODC, omithine decarboxylase; PBS, phosphate buffered saline, PC& perchlofic acid; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis Introduction Putrescine, spermidine and spermine are the most widely occurring polyamines in both plant and animal tissues. The involvement of these ubiquitous compounds in a wide array of cellular processes is extensively documented although their exact function is not understood (Flores et at 1988). Polyamines, which occur as cations at physiological pH bind ionically to negatively charged macromolecules including negatively charged phosphates of DNA, and perhaps Correspondence to: J. E. Carlson

influence the synthesis and transcription ofDNA (Feuerstein et al. 1991). Indeed, higher levels of polyamines and their biosynthetic enzymes are found to precede DNA synthesis during cell division (Minocha et al. 1991a, Amarasinghe and Carlson 1994). Evidence for involvement of polyamines in plant development include post translational modification of proteins by covalent conjugation to proteins via Wansglutaminase (Del Duca and Serafini-Fmcassini 1993), ~tion of hydroxy cinnamic amides of polyamines in organogenesis of plant reproductive organs (DeScenzo and Minocha 1993), and positive correlations of increased levels of polyamines and their biosynthetic enzymes during somatic embryogenesis (Galston and Flores 1991). In the well characterized somatic embryogenesis system of Daucus carota it has been established by a number of separate investigators that this developmental process is accompanied by increased polyamine levels and that arginine decarboxylase, a biosynthetic enzyme of putrescine is essential for embryogenesis (Montague etal. 1978, Minocha et al. 1991b). In spite of the extensive research into the role of polyamines in plant biology, investigations on the role of polyamines in gynmosperms has been very limited (Santanen and Simola 1992). In recent years considerable progress has been made in the clonal propagation of economically important conifers by somatic embryogenesis (Tautorus et al, 1991). In order to optimize mass propagation via somatic embryogenesis, proper characterization of this developmental process is of importance. In this context we have investigated the changes in polyamine biosynthesis during somatic embryogenesis in a cell line of interior spruce by qtvantification of putrescine, spermidine and spermine, as well as arginine decarboxylase and omithine decarboxylase levels.

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Materials and methods Plant m a t e r i a l The initialculturesof emlxyogenicrmsses of the interiorslmace(Picea glanca x Picea engelmamii cornplex)cell line W-70 were Ixovidedby 13.C. Research Inc. (Wcbsterctal. 1990). Theculmrcswcremairaainedat27~ inmladtmllighton

i/2 LM basalrmdium (Tremblay 1990) suppl~aontedwith 0.6% (w/v)Phytag~ BRL) ]% (w/v)ax~se, 10 p.M 2, * D (GibcoBRL} 5~tM~ a l n e

(~

(Si~.a), with wcckly mbcultufing. For further fftflh'eaiafionand malmation were lransf~d to hormone-free1/2 LM medknn wilh 0.6% (w/v) Phyagar, 1% (w/v) moroseand l%!~/v) activateddmra~ (Gn'bcoBRL) m x ~ a 16 h photolx~od (34 micromolesnf~s" ) for one week and lhon lransfetredto AE medkma (yon Arnoldand ~ 1981) with 0.63% (w/v) Phytagar,3.4% (w/v) ~ (w/v),40 a l ~ ' c acid (mixed isorne~s:Signa)and 1 pM indolebutyrieacid (Siffaa), under lhe same ligl~ c~adit~r~ For polyamine quanfificatioraand irmmmeassaysculture s~91es were collectedevery O.herday for 4 weeks,then immediatelyfrozen in fiquid N2 and storedat -70~ uxailassayed. Polyamlne qu~mfitkalions "Free" polyamims were extracted ilao 5% perdaloric acid (Fisher Scier~lic) mad after benzoylationwere qtmraifiedby revise phase HPLC using a Shin~zu CR 501 HPLC and a C18 reverse phase colunm (Shim-Pak ODS 5pro particle size and daection at 254 r~n) accusing to Flotes and Galston (1982). One halfofeach cultusesan'~le was fi'eeze-d6ed~ad used for olXahaingthe dry weigla. At each samplingtime, four replicates were ~ y z e d . The c~nplae expenment was cor~:baedin ~plicate,

Immtmoassay A time ~ for qtmr~cation of expressionof ADC ard ODC was cx~u~ct Total p r o ~ from cutatre ~luots fr~n each s~npling day were ~ in 100mMTrisatpH 7.2. "Ibeanxxmt ofprotein in each exlmawas quaraifiedand fl~eproteinbound to Polysorpinmxmo plates (Nunc Inc.) at 200/ag per well at 37~ ovemighL Plates were blocked~ 1% BSA in PBS, reacted wilh affinitypurified ar~'bodiesagainstADC or ODC at a titer of 1:400 for 3h at 37~ foUowedby reaclion wilh anti rabbit IgG-alkaline~ conjugate, p-nilmphenyt~ was used as lhe cbronaog~c mbgmle, and colous reaction was aopped atk~ 10 rain by ~he addifionof 1130gl 1MNaOH ardli~e colorimelrie~ were taken at 405 ma using aplale reader.

Results Somatic embryogenesis When the W-70 cell line was cultured in maintenance medium containing 2,4-D and BA, it grew as dense masses of rapidly dividing ceils at the culture periphery with proximal non-dividing suspensor-like elongated cells. After transfer to differentiation medium lacking hormones the culture ceased rapid growth in mass but differentiated further to produce bilaterally symmetrical, ~ent early somatic embryos which continued to increase

R,~s~ngpolydonal antibodies a g a ~ ADC and ODC ~ y available Escherichia coli wginine dacarboxylx~ and c~r~t/hine decarboxylase (Sigma) were fuaher purified by SDS-PAGE. Bands con'estx~adingto molecularmass of72 kD for ADC and 81 kD for ODC were excised (Morris and Bod~er 1983) and the proteins eltaed and ~ a l e d using an eleclmeluter(Amicon Coalrilutor)at 4~ Rabbits were itr~nu~xt with 100gg of flaearfigan ADC or ODC, mLxed1:1 with omapleteFretmdsac~uvara,and followedin two weeks by two boos~ hajectionsa forlnightapart At~iserawere collected10 days after the ~ ~ injeaiort Preinmatmizationserum samples were also collected fiom eada rabbit At~ly ~

ofpolydonal m~tibodiesand western b l o t ~ g Polyclonalaraiseraagah~ ADC madODC were affinitypurifiedby an adaptationoflhe ~ desmbed by P~anen (1992). Briefly,E. coilADC and ODC were fractionated by Q Sephar~e ~tfion exchan~ d ~ , ' ~ l y , fractions coneslxxding to major protein peaks v~re run on SDS-PAGE and bands to 72kD (A]X3) or 81kD (O13(3) were transfened to nitrocellulose paper by elearoblotting, Memlxane stripscoraainingfile blottedproteinwere blocked with 5% BSAin PBS for 1 h at room tarOwaltne, hxadxaedwith the am-espotding az~ise~annfor 12 h at 4~ and washed willi sevca-alchanges of PBS. "1hecaptured ar~'bodies were eluted wifffin30 s with 400/al of elution lxtfl'er containing 5raM glycineat pH 2:3, 400raM NaCI mad0.2% BSA and inmaedialelymtamlizedwifl120 pl 1MNa#~phatebuffer, pH 7.0. Theeluted m~ilxxlieswere dialyzeda~-Jst fonr dmnges ofPBS over 12h. Tolal proteinexlractsofthree day old mah~enancecultures(preinduction emlxyogenicmasses)were run on 10~ a~2clamideSDS-PAGE and non-demawing PAGE (SDS and 13-mercal~oeflaanolomitted),eleclroblolled,and reactedwith eitherof the two affinitypurifiedaraJbodiesagainst ADC and ODC at fltersof 1:400 followed by detectionwith arai iabbit IgG-alkaline pheqtmle conjugate u s ~ the Prornega westernblot kit. For SDS-PAGEboth boiledmadmlboiled~ exlraclswere used. Cornels were perforrmd either ~ t ~ the lairrary ~"oody or using r ~ o n serumin placeofthe The ideraity of the bands recognized by the two araibodies were c~tfitrmd as follow~ Total protein of flree day old m a i r ~ a c e culawes were exlracted in 100 mM Tris bt~t'erpH 7.2 and were fractiomted by anion exd~ange dromatogaptry using a Bio-Rad Econocoltum syaem ~ a Q sepl~n~seanion exchangecolttrl~x 100 ~1 samples office ct~natoffaplgr fractionswere dot blolted two dot blotswere probed separatelyushagfl~etwo aff~ty ptn~ed araibodiesand lhe Paxnega ~ blot de~x~onkit The fractions~fiida were identifiedby flaetwo argibodieswere Ix~oledand eoncea~aed separatelyand ADC or OI)(2 activitywas assayed using l-14,-Carginineorl- 14Congthine, adq~fl~emetlxaddescrlbedby Robie and Minocha (1989). Enz'yn~ assays were also perfom~d for lhe crude total proteinexWacL ~ l s were performedusing droffmtogr~ay fi'actions'Mfidlwere not~ by~e am~oodiesandby or~ing tl~ d r c ~ fra~m

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maintenance I

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differentiationI Time (days)

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F'~me 1. Quantification of 5% perchloric acid soluble polyamims during s~natie

emtryog~sis in ir~or spruo~. ~

(D~ Spenni&~e(O1 Spetmire(a).

phase: 2,4 D and BA supplemented I/2 LM med&mawith enCJyoge~ tissue, differelaiation phase: H ~ a ~ l e free 1/2 LM medium wilh early embryo differeraiatiort mattualion phase: ABA and IBA supplemeraed AE medium with

~ o n

ofsomaticcmtxyo~ Arrows indicatelime oflrarffcrto d i f f ~ o n

ard

maturation media. Each value is lhe nx~n (:/~qE) of four replicates and the vahJes

in size by cell division. When cultures were transferred to the maturation medium supplemented with 40pM ABA and lp_M IBA the embryos continued to grow in size, and within 2-3 days on ABA embryos started to elongate and became opaque and yellow indicating protein and lipid synthesis that a c e o ~ e s maturation. At the end of 2 weeks following transfer to the maturation medium, most embryos were at the cotyledonary stage.

497 protein. Interestingly, if the protein was not boiled with loading buffer prior to loading the gel this band occurred as a doublet on the western blot. A similar doublet was also seen on the nondenaturing westems of total protein when anti ODC anU2xxiy was used. In the control western blots which were reacted with the preimmunization sera, several faint non-specific bands which do not correspond to the bands recognized by the antiADC or anli-ODC antibodies appear (Fig. 2). These background bands are much reduced on the westem blots detectedby the affinitypurified an/a~xlies.

Quantification of PCA soluble polyamine levels Of the three polyamines, putresdne levels remained the highest throughout the time course study (Fig. 1). On the maintenance medium, the highest level of putrescine was observed five days after transfer, which also coindded with the rapid growth phase of the embryogenic tissue. A more dramatic two fold increase in putresdne level was seen six days after transfer to the maturation media, corresponding to the time when the embryos reached the globular stage. This high level of putresdne, however, dropped off to predifferentiation levels within five days. Spermidine levels which decreased in the differentiationmedium lacking hormones, continued to increase after transfer to the maturation mediun~ Maximum spermidine levels lagged two days behind the putrescine peaE Spermine was the least abundant of the three polyamines, and changed in a pattern similar to spermidine. The pattern of change of the three polyamines was the same in replicate experiments.

Correlation of enzyme activity and antigenicity of protein b,'mds Anti- ADC ant~xXlydetected one broad peak among anion exchange column fractions of total callus protein when the fractions were dot blot immunoassayed. The fractions corresponding to this peak were concentrated and when assayed for enzyme activity using 1'14C arginine showed much higher specific activity for ADC than either total crude protein or similar quantifies of protein from fractions which were not recognized by the antllxxly. The same procedure when repeated for anti-ODC matibody showed one major peak and the corresponding fractions also had higher levels of ODC activity relative to total protein or to the fractions not recognized by the antibody (data not shown). The correlation of sqxcificenzyme activity with recognition by the corresponding anlaqxxlies confirmed the specificityof the antlqxxliesto the two enzymes. 0.7

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8 .~ 0.3 Figure 2, Coc~assie stain of ixotein f r ~ n iraerior spruce m t r y o g e n i c tissue and western blot analysk Total protein exlracted from caUtts (100Dg per lane) was by SDS-PAGE (A-F) or native PAGE (G), I m r f f m e d to tfilrocellulose and

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wi~polycl~alamtxxtiesagairaADCandOIX;. A: Molecular weight ~ ( ~ minlx)w marker), B: ~ e stained total soluble praein from embryogenic fisa~. C, D, E: SDS-PAGE bli:~s probed with arai-

ODC(C),arai-ADC(I))andtxe-kr~nunesenzn(E). F: sameas C, butwithout boilingofsm-Jplebeforeloading. G:.non-denattwingPAGEblotprobedwithantioIx: am'o,~y

0.1

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9 ' .... 3 5 maintenance

Polyclonal antibodies against ADC and ODC The affinity purified polyclonal anlflxxties raised against E. coil ADC recognized a single band corresponding to molecular mass of 65 kD on SDS-PAGE western blots of total protein isolated from spruce embryogenic lissue (Fig. 2). This band was absent from the westem blots of non-denaturing gels. The anlfixxlies raised against E. coil ODC recognized a single band at 85kD on SDS-PAGE western blots of total spruce

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, r , i , i , i , i , r , i . i . , . , 9 1I 13 15 17 19 21 23 25 27 differentiation

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F ' ~ m e 3. ~ t a x ~ a s s a y o f A D C ([]) and O D C (O) during sciatic emlxyogmesis in irlericcspmce~ ~n'haglhesame e ~ as in figure 1. T ~ a l txoleinwas boundto

hrmunoplates(200gg perwell)andthe enzymeswerequnr/ifiedusingamlxxties agair~E.coliADCandODC. Eachwalueislhemean@SE)offourreplicatesfrom oneext~nenL

Immunoassay Throughout the time course experiment ADC levels remained

498 higher than ODC levels. When cultures were transferred to hormone free differentiation mediunL the ADC levels remained constant (Fig. 3). On maturation meditun, ADC levels increased more than two fold, reaching a peak nine days after transfer, and remained high during the rest of the experiment. ODC levels on the other hand did not show any significant change correlating with maturation of the somatic embryos. A duplicate experiment showed the same pattern of change in ADC and ODC levels, except for a slight reduction in ADC levels in the hormone free mediurrL Discussion The association of polyamine biosynthesis with somatic embryogenesis was first demonstrated for somatic embryogenesis in carrot (Montague et al. 1978). In carrot, a two fold increase in putrescine accompanied by increasing ADC activity was found to be associated with embryogenesis. These obsen~ous were later corroborated by the use of the biosynthetic intffoitors difluoro methyl arginine and difluoro methyl ornithine, which inhibit ADC and ODC activity (Fierer et al. 1984, Robie and Minocha 1989, Minocha et al. 1991b). Of these, the last two reports indicated that spermidine is more linked to embryogenesis than putrescine and Minocha et al. (1991b) suggested that this is due to the reduction of ethylene synthesis in tissues undergoing high spermidine biosynthesis since both these compounds share the rome precursor, Sadenosyl methionine. This general pattern of association of polyamines with somatic embryogenesis in carrot, as well as in several other plant systems (Galston and Flores 1991), is consistent with our data on "free" (PCA soluble) polyamines and ADC and ODC levels during somatic embryogenesis in interior spruce. We observed that putrescine levels increased two fold during the embryo maturation process reaching a peak coinciding with the differentiation of early embryos to globular embryos, but decreased to original levels thereafter. Increase in spermidine levels was also seen during the embryo maturation process, with maximum spermidine levels observed just after the putrescine peak, likely illustrating the conversion of putrescine to spermidine. These results differ considerably from the results reported by Santanen and Simola (1992) for somatic embryogenesis in Picea abies. They observed a dramatic 10fold decrease of putrescine inamediately after transfer to maturation media. However, they also reported flmt spermidine levels increased in association with the maturation process. The high level of ADC persisted even after putrescine levels had dropped off to the predifferentiation levels in interior spruce cultttres could reflect rapid conversion of putrescine to spermidine or conversion of "free" putrescine to conjugated forms such as hydroxycinnamic derivatives or protein conjugates, which are implicated in developmental processes. The relatively low and stable ODC levels observed during embryogenesis in interior spruce show similarity to other cell systems (Robie and Minocha 1989). The high levels of

putrescine seen throughout the experiment likely reflects rapid cell division, as previously reported for other fast pro"lffemting cell systems (Minocha et al. 1991a). The present study investigated the levels of ADC and ODC during somatic embryogenesis by immunoassay. Although immunoassay may not directly correspond to specific activity of the enzyme due to possible post translational modifications or end product inhibition, it nevertheless gives an accurate quantification of the amount of the enzyme present. Immunoassay is not affected by artifacts such as loss of enzyme activity during extraction or artificially elevated levels of activity due to non-sl:edfic release of CO2 from the substmte. Nonspecific release of CO2 could be due to diamine oxidase activity which is likely to be present in the crude protein extract, or could be due to the presence of phenolic compounds leading to non-oxidative decarboxylafion of the substmte. The combined action of arginase and urease can also cause rek~se of CO2 from arginine (reviewed by Birecka 1991). Immunoassay is also not prone to technical variability which is imminent in CO2 capture enzyme activity assays which can only be overcome by the use of a very large number of replicates, which may not always be practical. In the present investigation we found spruce ADC to have a molecular mass of 65 kD, which is similar to ADC of oat reported by Mahnberg et al. (1992). However, spruce ADC appeared as a single band on SDS-PAGE western blots under reducing conditions (with B-mercaptoethanol) and did not show evidence of post translational modification as reported for ADC of oat. Several plant ADCs had been found to be mul"tLmeric (Smith 1979, Choudhufi and Ghosh 1982) and our inability to detect spruce ADC on non-denaturing PAGE western blots suggests that this ADC may be a large multimer which fails to enter the gel. Spruce ODC was found to have a molecular mass of 85 kD and seems to be present as two isoforms as detected on western blots under non-denaturing conditions. By exploiting the relatively high level of homology between E. coli decarboxylases and the respective enzymes from higher organisms (Rastogi et al. 1993) we were able to produce polyclonal antibodies against ADC and ODC using commercially available E. coli enzymes after a single purification step. These two antibodies which specifically recognize spruce ADC and ODC on westem blots could provide a valuable tool for purifying these enzymes from other plants and also ha investigations on the regulation of polyamine biosynthesis in other plant systems. The results from quantification of putrescine, spermidine and spermine levels mad ADC and ODC fiters show that a correlation exists between polyamine biosynthesis and somatic embryogenesis in interior spruce, similar to embryogenesis in other plant systems. During embryo maturation the putrescine and spermidine levels and ADC titers increase above those found in rapidly dividing undifferentiated callus cells indicating that higher polyamine levels are associated with differentiation and maturation.

499

Acknowledgements We gatefully admow!edge Dr. D. 1L Robe~ Ms. Fkma Wehaer and Ms. Stephanie McGukess of B. C. Research Inc. for lhe provision of embryogenicculturesofirmior spruce, and Pros NeLlTowers ofthe of Bolzny, University of British Coltmabiafor accessto lhe HPLC facility. This research was ~ by, Nattral Sder~e ~ad FaagineerlngResearch Ceuncll Researda Grar~numberOGP0~ 1724 to J.E.C.

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von Arnold S, ~ T (1981) In vitro stu~es on advenfitioossh:~t formation in t~us co~x~aa.C2m.J. Bot. 59:870-874 Webster FB, Roberts DR, Mcirmis SM, Sutton I3CS (1990) Propagation of kaerior stxuce by sonaaficen~ C_xatJ. For. Res. 20:1759-1765