Plant Plhvsiol. (1967) 42, 1587-1595 Gluconeogenesis from Amino Acids in Germinating Castor Bean Endosperm and its Role in Transport to the Embryo1 Cecil R. Stewart2 and Harry Beevers Department of Bio!ogical Sciences, Purdue University, Lafayette, Indiana 47907 Received July 5, 1967. Sutm mslary. During germinlation of the castor bean all of the conitenlts of the endosperm are ultimately transported to the enLbryo through the cotyledon or respired. A net loss of nitrogen from the endosperm begins about the fourth day, i.e. at the time wlhen embryo growth and fat breakdowni are also beginning. Amino acid analysis of the exudate from the cotyledons, still enclosed in the endosperm, shorwed that the amounts of aspartate, glutamate, glycine, and alanilne 'were very low and that glutamine made up 40 % of the amino acids in the exudate. Amino acids laibe'ed w-ith 14C x-ere applied to intact excised endospermiis to follow utilization. Aspartate. glutamiate. a'anine, glycine, cer'ne, anid leucine were converted to sugar to varying extents. Proline, arginine, valine, and phenylalanine were not appreciably converted to sugars. Proline and glutamnate were converted to glutamine. When 14C-glutamate, aspartate. and alanine were added to the outer endosperm of intact 'zeedlings, onl,y sugars and glhitamine contained appreciable label in the exudate. When 14C-valine was added, it was virtually the only labeled compound in the exudate. The results show that anmino acids iwlhich on deamination can give rise to intermll2diates in the pathlway of conversioin of fat to sucrose are largely converted to sucrose and the nitrogen transported as glutamiine. Other amino acids released from the endosiperm protein are transported intact into the seedling axis. Some carbon from the gluconeogenic aimino acids is also transported as glutamine. In fatty seeds such as those of the castor bean the storage reserves are present in the endospermii tissue surrounding the end. ryo proper. Dturing germination the major bioclhetmical event in the endospermii is the massive conversion of fat to carbohydrate (3) which is then absorbed tlhrough the cotyledons of the groxv- ing embryo (9). Now although fat is by far the major reserve in the endosperm, proteins are also present, and in fact synthesis of particular proteins from soluble precurs!ors occurs early in germination (1). However, after a germination period of 8 to 10 days essentially all of the material originally present in the endosperm. including the protein, has disap- peared, and only the papery outer integument remains. The fate of the protein component is thle subject of this paper. The breakdown of protein duringi germiniatecn and aspects of transport anid utilization of the produicts in the embryo proper have previously been exmiined (6), particularly in legumes (11) and in cereal seed- lingl (7, 15, 16). From this work it w as anticipated that some of the amino acids produced during hyidrol- ysis of proteini in the castor bean endo-permi would be I Supported bw- the Utnited States Atomilic Energy Commission (AT-11-1 330). - N.I.H. Postdoctoral Fellow. transferred directly to the embryo, but that mietabolic transformations such as oxidation and tranisamiiinatlon would also intervene in the storage tissue. It soon became apparent that, in addition to these events, a process which in other types of seedlings (7, 11. 16), and indeed in most other plant tissues (8, 17), is of marginal significance, here assulmies great importance. The carbon skeletons of some of the aminl,o acidls wvere converted to sugars with remarkable efficiency. Gluconeogenesis from amino acids is of course well documenited in particular animal tissues (22). The points of emphasis here are the scale of the convers:on in the castor ibean endosiperm and its contrilbution to the transnmittal of carbon from protein in the endo- sperm to the growing embryo. Materials and Methods Castor beans, Ricinits comimm is L. (var. Cim- maron) 'were germinated in vermiculite at 30' after soaking in water for 8 to 12 hours. Labeled Substrates. '4C labeled aminio acids were obtained from commercial sources. All except serine were uniformly labeled, with specific activities from 60 to 200 mc/mmole. Serine-3-1AC (4 mc/mmole) was used. y-Aminobutyrate-U-'-C was prepared by incubating glutamate-U-1 'C with commliercial glutamic 1587 Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
Transcript
Plant Plhvsiol. (1967) 42, 1587-1595
Gluconeogenesis from Amino Acids in Germinating Castor BeanEndosperm and its Role in Transport to the Embryo1
Cecil R. Stewart2 and Harry BeeversDepartment of Bio!ogical Sciences, Purdue University, Lafayette, Indiana 47907
Received July 5, 1967.
Sutm mslary. During germinlation of the castor bean all of the conitenlts of the endospermare ultimately transported to the enLbryo through the cotyledon or respired. A net lossof nitrogen from the endosperm begins about the fourth day, i.e. at the time wlhen embryogrowth and fat breakdowni are also beginning. Amino acid analysis of the exudate fromthe cotyledons, still enclosed in the endosperm, shorwed that the amounts of aspartate,glutamate, glycine, and alanilne 'were very low and that glutamine made up 40 % of theamino acids in the exudate.
Amino acids laibe'ed w-ith 14C x-ere applied to intact excised endospermiis to followutilization. Aspartate. glutamiate. a'anine, glycine, cer'ne, anid leucine were convertedto sugar to varying extents. Proline, arginine, valine, and phenylalanine were notappreciably converted to sugars. Proline and glutamnate were converted to glutamine.When 14C-glutamate, aspartate. and alanine were added to the outer endosperm of intact'zeedlings, onl,y sugars and glhitamine contained appreciable label in the exudate. When14C-valine was added, it was virtually the only labeled compound in the exudate.
The results show that anmino acids iwlhich on deamination can give rise to intermll2diatesin the pathlway of conversioin of fat to sucrose are largely converted to sucrose and thenitrogen transported as glutamiine. Other amino acids released from the endosipermprotein are transported intact into the seedling axis. Some carbon from the gluconeogenicaimino acids is also transported as glutamine.
In fatty seeds such as those of the castor bean thestorage reserves are present in the endospermii tissuesurrounding the end. ryo proper. Dturing germinationthe major bioclhetmical event in the endospermii is themassive conversion of fat to carbohydrate (3) whichis then absorbed tlhrough the cotyledons of the groxv-ing embryo (9). Now although fat is by far themajor reserve in the endosperm, proteins are alsopresent, and in fact synthesis of particular proteinsfrom soluble precurs!ors occurs early in germination(1). However, after a germination period of 8 to10 days essentially all of the material originally presentin the endosperm. including the protein, has disap-peared, and only the papery outer integument remains.The fate of the protein component is thle subject ofthis paper.
The breakdown of protein duringi germiniatecn andaspects of transport anid utilization of the produicts inthe embryo proper have previously been exmiined(6), particularly in legumes (11) and in cereal seed-lingl (7, 15, 16). From this work it w as anticipatedthat some of the amino acids produced during hyidrol-ysis of proteini in the castor bean endo-permi would be
I Supported bw- the Utnited States Atomilic EnergyCommission (AT-11-1 330).
- N.I.H. Postdoctoral Fellow.
transferred directly to the embryo, but that mietabolictransformations such as oxidation and tranisamiiinatlonwould also intervene in the storage tissue. It soonbecame apparent that, in addition to these events, aprocess which in other types of seedlings (7, 11. 16),and indeed in most other plant tissues (8, 17), is ofmarginal significance, here assulmies great importance.The carbon skeletons of some of the aminl,o acidls wvereconverted to sugars with remarkable efficiency.Gluconeogenesis from amino acids is of course welldocumenited in particular animal tissues (22). Thepoints of emphasis here are the scale of the convers:onin the castor ibean endosiperm and its contrilbution tothe transnmittal of carbon from protein in the endo-sperm to the growing embryo.
Materials and Methods
Castor beans, Ricinits comimmis L. (var. Cim-maron) 'were germinated in vermiculite at 30' aftersoaking in water for 8 to 12 hours.
Labeled Substrates. '4C labeled aminio acids wereobtained from commercial sources. All except serinewere uniformly labeled, with specific activities from60 to 200 mc/mmole. Serine-3-1AC (4 mc/mmole)was used. y-Aminobutyrate-U-'-C was prepared byincubating glutamate-U-1 'C with commliercial glutamic
1587
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1588 PLANT PHYSIOLOGY
clecarboxvlase andI purifying th,,e produict by paperclhronatography.
Determination of thec Nitrogenoll sCComponents(tabe I ndf igI) ). Ten seedlingswr-ere harvesteddaily. The vermiculite wvas removed as carefully aspossible to prevent loss of root tissue and
lingis separated ilto endosperm anid emibiryo w liichixerckilled by immersion in boiling 95 % ethianol. ThettISSLie was, then gerouind in aVirrtis homnogenizer andthorotighiv eXtracted with 80 %ethtanoll 1w repeatedcentrifug-ation andl resuspension. Theol alcohool extractxvas evaporated to dlrynessC at <400 anld takenUp in
xv-ater after remiovin-g lipids w ithchlloroformii.Alphla-am-ino nitrogen xvas dletermnedlon1 the xxwater extract(12). The residueAwNasdriedc andhlvdrolyzed in a
sealcd tube contaiining1 1:1 glacial acetic acdd:3 N
HCl for 16 to 18 hours at 1200. Alph-atamnnoniitrooeni was dleterminiede oni the hvdrolN sat!s (12)anidtlils xxwa used as a mieasuire of Protein n:trogenfig1 A) Theamliniio acid cmipostion of the 'ndo-sperm proteiinwxas determined by anavzing the resduehN-drolvsate froml-tin-ermiiinated seeds with a T1'ecl-nlicon1 Autoalnalyzer.
Collect1ion andlnalysis of1.itxdate. Thtc hvypo-cotvlI of5f davs(old seedllinosx-cere excis,-d co thatabout 3mmll of thelhypocotyl rema:ned heloxw thecotxledlons xxhich wxere still enclh>,ed in the encdosperm(see fig 3 in refere-nce 9). TI'hiis 3 mim section wasinisel-ted( into a snug-ly fitting glass tulbe anidtlhl seedspiaced inamloi. t atmioslphere.ExTuidatexx-as collectedfrolm sex-eral seedlings for 4 to 6hours. For anminoacid analysis,thl exutdatexxas passedtlhrougl h DoNvex-1-forni-ate to remiove glutarmate alnd aspartate. Tleceffluenit xxas hvdrol\ zedxxth1 N HClcat 100 for2 hours. This hcirolvsate xvas examiiniedI wx-th an
am no ac.d anaal-zer. Glutamiate and aspartatexNveredetermiiined separatclv xvith the alnalIzer afterelutlt onfromii Dowex-1-forni-ate xvithl 4 N formnic aci,d.
In the experimients inxlvich 1-(C in the extudatevas analyzed, the exuidate.s wxere fractIonated into
amiino ac;d, organic acid, and non-ionic fractions bypassing thelm succe_ss:vevly tlhrouglh Dox\vcx-50-H+ andlDoUvex-l-forimate. Amino ac.ds xx ere eluted fromDowxex-50 xvith 2 \ NH,OH and organic acids xvereelditecl fromn Do\xex-l-formate xxith 4 N form:c acid.
Adminiistration of Labele( Ahnino .4ci(ls to the Ex-cise( Endospecrni (figs 2-10). Enndospermihalves vereremoved fromi 5S da-s o0l seedlings. For each treat-iiemit, 4 endouspcerni hal\ es xere uscd anldl approxi-niately 0.5 uc of 1IC labeled ami.no acid (0.005-002/mole in 10-20 AX) xwas applied to eaclh. ThFe radio-actixve solutionl xxas spread directly cl-i the inner sturfaceof the endospermi pieces. Dtue to the ability of endo-spermii to absorl) xater, the radioactive solution xvasrapidly absor-bed ('15 mins). The ti-sue xas p'acedin. a closed 5)00 mil Erlenme\ver flask containing asmall amnount of xxater to provide a nioK.t atmosphere.A separate sample wvas incubated for each timie oftreatment. In s;omie of the exDcriimelnts. CO., wascollected by suispending in the flask a 2.5 X 2.5 cmpiece of filter (paper saturate(l x-itlh 10 % CO-free
NaGH. In the reiiainingY cxperiihocits, a scintillationvial containing 10 drops ofhyyaninie and 10 d1rops ofwa9ter xas placed in the closed flasks.Th'he 2t etoods,gavecompparabbe results and a qulant:'tativxerec(-vervof 14CCO. xvas observed xvhen a kinowvn amounit wvasreleased into the flask byinject.ing acidintto a14Cbicarbonate solution. At thecnd of theincCL1cbation,scintillation solution was added to the filter paper orhvamine in a scintillation vial and thett conitentdetermined. At the -end of the incubaton, tie tssuewas killed by immersion in boiling 95 % cthanol,extracte(l, and fractionated inito aminoacl, l or anicacid, andI non- onic fractions as described( abox-c TIminsoluble residte duvas discarded. Aliquiots xvereassayee-d for 14C on a scintillation counter. In eachiexperimlenet the total 14C recovered at eachi sampnlingl.ihichl included the tnmetabolized ami:no acid, remainedessentiallv constant (2 XWI' cpm). The dato pr-e-sentedlshow the amiount of' 4(C in eachlfiaat'opercent of the total radl oactivitxreecoxc red.
Paper Chromiratography of.Jniiino Acids modSugars. For thedcteriiniation of the d'str:lliuttonofra(lioactixitv- in the aniino acid fraction.thle aminonac:ds xvere separatedby 2-dimensional pap2r chronia-tography((20). 'flhe solxenit tise(l inlb firi st(Icre-tion xvas n-butano! :acetic acid:xx-ater 1 2:3 :5) ani(lin the secoind direction plhenol:x-xater (80 :28). \fterchromatographythe amino acid, xwere locatedb1 theirfluolrescenice under LTV lightin the pre-ence of1,2-naphltlhoqtminone-4-silfoniate ('13), the si(Iots; xereexcised, cutup, and placed inscintillation vials. TWOml of xxater were aclde(d alnd after 1houlr sciutTationsolutionxxas added andl the 1WColtnlltent Of til samiplesvas measuired.
The non-ionic fractions x\x-rc chromnatographedaccording to theimietlhodl described bx Kriedemann andBeevers 10) Small aliquot- xxwere streaked oniW"Ihatmiian 3 MM paper and after chromatography inethylacetate:pyridne:xater ( 10:4:3) the suigars\xverlocatedlb)y sprayng Imarker strtps, with amoniunmolvidclate reagent (2). Regions of the paper con-tamning suticrose, glucose5, anid frictose wvere tlheni cutout, Lhe sugars \xvere eluited andi tlhe rad1oact;xx txdeterminied.
Results
Chlaniges int Proteinia1(l Solibie-N Contcet I)nringGernmination. Under the conditions emplo ed, theendosperm is comp'etely digested 1y the telntlh (day' ofgermination, xvhen the radicle is 1(0 to 15 cm in'engthl.Faclh -Ced contais in:tiallv about 300 mig of fat andrapid breakdown begins oni the fouirtlh day of germi-nationl. From the data in fiigutre 1 it is seen thlat theprotein content of the ungerminiate(l seed is abotit40 mg (6.25 X the protein amino-nitrog-en contenitfrom fig IA) and that a rapid nelt los., Occurs froimthe endosipermii beginning also at day 4, despvite thlefact that enizymne synthlesis i;, occurring at this tilne(1). The loss of proteini froium the endosperm :sreci,procally related to a gaini of protein in the eniirvo0
15Xt8
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STEWART AND BEEVERS-GLUCONEOGENESIS FROM AMINO ACIDS
c
06
C,oE 6 EmbryoA
z 6
niroe (B*E h ndospermanemrofcsr
00~
0
r m a
I- z
2.0-J
FI.O 1.6 Cagsi rti A n oul mn
and the soluble amlino-N content of the emnbry-o (fig1B) also increases from day 4. The growth oyf theembryo may,provide a continuous silgnal for proteindegradation. in the endosperm, although as seen infigure 1lB there is a temporary accumulation ofsoluable N-compounds in the endosperm itself.
Nitrogenous Constituents in the Exuldate frontzCotyledons. By severing the hypocotyl just below thecotyledonary node, exudate can be collected whichcontains material in transit from en,dosperm to themainl emobryonic axis (9). The amino acid composi-tion of this exudate is show-n in table I. wshere it iscomparedl to that of endosperm protein. A strikingfeature of the ex;udate is the virtual absence ofglutamnate and aspartate and the low- amounts ofglycine and alanline compared to those in the endo-spermn protein. These almino acids are of courseclosely related to intermediates in the conversion offat to sugar (3). Glutamine comprises 40 % of theamino compoundls in the exudate. It is clear then
that the overall amino acid composition of the bulkendosperm protein is not reflected in the solubleconstituents derived from it and moving into thegrowing embryo. The dominance of glutamine andthe disproportionately low amounts of particular aminoacids in the exudate presumably reflect an extensivemetabolism of hydrolysis products in the endospermitself.
Thle Fate of Labeled Amttinto Acids in ExcisedEndosperm Tissuie. In a series of ex.periments indi-vidual labeled amino acids were provided to excisedendosperm halves and samples taken at intervals overa 12 hour period. The incorporation of 14C intoCO, nonw-ionic, organic acid, and amino acid fractions
Table I. Antino Acid Aialysis of Exuidate fro 1tHypocotyls and of Enidospermn Protein
* Total ammonium in exudate after hydrolysis minusglutamine and asparagine.
** Includes corresponding amides.
was measured. From those amino acids closely re-lated to intermediates in the pathway of conversionof acetate to sugars there was a remarkably efficientincorporation into the non-ionic fraction (figs 2-7).When this occurred it was shown that the 14C wasin fact in sugars and the data in table II, includingthe effects of invertase, establish that sucrose is theprincipal product, with roughly equal amounts of 14Cin its glucose and fructose moieties. Smaller butequal amounts of 1+C twere recovered in free fructoseand glucose. This is the pattern which was observedearlier in experiments with acetate-14C (5). Theresults obtained with particular amino acids are de-scribed below-.
Aspartate (fig 2). More than 50 % of the addedaspartate wras metabolized during the first hour and
1589
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Table II. Aloalysis of t1/e Neutral Fraction after, Ado iistratio,l of I4C-am(/ino . It id.t
Radioactivity per samlp)lewvithoutit vertasc pretreatmentSu1cr-ose Glticose Fruciose
chroinlatograplieclpretreate(l xx-itli invxertase
Sultcrose (;G11coe Fruct >c
AIalillI i - L'- CAsparilatc-U' 'IC
Seriuci -3- 4.cGilutaulatcU)j l-IC-y-Aminol)tiyrate- c
there was a striking incorporation ot ' inlto organic
acids. After tllis timiie. as the in organic acidsdeclined, there wx-as a linear increase of 14C) in sugars
and a slow release of 4N O._. Fractionation of theorganic acid fraction at 2 hours howed that 90 %
of the ,' w\ as in malate; fumiiarate containedimostof the remaining 14C in tllis fractionl and succinateand citrate wxer- virtuallv uinlaheled. C'hr-omato--raphvof the amino azid fractioni at 12 hour,; showved thatalmnost all of the 4C remaine,d in aslpartate, therewas no conversion to asparagine.
Gliutaiatc (fig T).The utilization of a(lded glu-taimiate wvas somewhat more complex. 'More than28 % of the '' C recovered after 12 houirs was insugars anid less than 4 % in CO_. However therewas aln extensive conversion to g,lutamine, w!hiclaccounited for 20 % of the 4{C recoverecd at 3 hours.
an,d (Itdring this tinme also there was a buildup of
labeled y-aminobutyrate. 'rlTe fact that '4C iny-aminobutyrate subsequently declined suggested thatitmav be an intermediate in the conversion of gluta-mate to sugars.
y-Anxinobutvrate -\ as uitilized less readily thanglutamate but conversion to su,gars di(d in fact occur
(fig- 4). It appears that part of the glutamate isconverted to sugar thlroughl a path,way beginning xvitha dlecarboxylation step. However, the results do notallowv a conclusion as to what fraction of the addedglutamate is metabolize,d throughl a-ketoglutarate.
Alaniine was con,verted to sugars more efficientlxthan anv of the acids studied (fig 5). -Morc thlan60 % of the 1 1C recovered after 12 hiours was presentin the sugar fractioni anid tlhere vas only a minor-inicorporation into organic acids. Oni the blts:s of
previous experiment. with endosp2rnm slices in wiich
l)yru-\a;te utilization was shlo-wn to occuir b1 w-ax ofthe gl-oxvlate cyc'.e after conversion to acetvl CoA(14), it was inot anticipatell that ilmore than 50 % oftlle alanine carbon w-ould be recovere(l a.s sugars.
'The superior conversion observed in the pr\ sent ex-
periments in wx hich anialinie was appli(l directly toendosperimi halves is probably due to a miiore efficientrefixation of 1 CO., anid its uitilization in sutiar syn-
thlesis (4, 19). This interpretation is supported bythe observation that w7h1ien pyruvate-2-1 4C and ace-
tate_I4-C vel-e applied 1x- the techllnqtle.; used in the
present series ot experiments, greater recoveries otxvCNN-ere obtained in sugars than xvere obser-ved in
the previotus experiments using slices ilnctibatedl insolution.
Glycine alnd serine xvere botlh colnerted to llugars;more than 30 % of the ',(- recovered after 12 hours
was in this fractioln (figs 6 andl 7). Thle realdy conl-version of gl-cine mnay reasonably be ascribed to itsrole as a precursor of glyoxylate. In th'e experimiienltshown in figure 7, serine-3-'lC' was uied. 'The factthat C'-3 of serine xvas a good sugar l)recnrsor incl-cates that serine xvas not first converted to glxvcine
and glyoxylate. fransaminationi to hydroxypyruxvateand direct utilization of a 3 carbon uiinit in Stigarsynthesis is suggested.
Leucinie wvas utilized slowvl\ it as the only
labeled coimipoundI in the aminio ac.d fraction xvhiclhcontained, anlmost 80 % of the 4C' present after 12hours (fig 8) Howx'ver, althou,Th only marginalamiiounts of 14C ere recoxered in (O, anid organiicacids, there ,vas a progresslive appearance of 14 lli
sugars during the exiperiment; as for the otlhe, sugiar
precursors. sucrose xvas the major product.Gluttamiiine was also slotvlitilized; it wa.s con-
-ertecl to sugars less readily tlhan x as glutanmate(fig 9)). ihromategraphv of the aminio aci(d fractionafter 12 hours showed that 10 % of the 14(' w-aspresent in glutamiate, 'withl no significant labelincy in
any other amino acid except gtlutami.ne. T]his be-havior is consistelnt witlh the role of glutamine as a
major sink and transport formii for amilino groups.Proline, argininne, valine. and phenylalan.-ne are
grotuqped togethler because they are all utilizecl i-ervs!owvlv, and, Nxxith the exceptioln of proline, virtuallynot at all for sugar ;vntllesis (sfig' 10). The 14CO.0Otitl)ut is niot shovn; in none of thlese experiimeintsdid it contain mnore thani 0.1 % of the 14{C recoveredafter 12 hours. 'rhe organic acid fraction wvas alsover\ feebl- labeled. Tllhe anmino acid fractions rie-
covered fromii each of the experimen,ts shovnn infigure 10 xere examined in the automatic anialyzeranfl the 14C determined oln a flox scanner. In tlleamino aci(d fraction from the proline-l-C expernimenit85 % of the w'Cxas presenit as proline, 10 % wvas inglutamine and5 % in glutamate. The slow couxner-
Sionl to glultamate probablx accounts for the fact that
Ainino (Ici(Iackde 4
90/)8f)0
190)2,500900
140(150030)
cI)III250))49002300490034003007091100600
C'l)In13.1009000300059()04500)7000340014001000
cp)m1112,700)9000360059)00590(0790037001700700)
1590 PLAiN-T PHY\SI0tOLOGY
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1591STEWN ART AND BEEVERS-GLUCONEOGENESIS FRO'M AMINO ACIDS
a
whI
'0
U
hi
-i4~-.0I-
II
0
49
U)z
0
49U,
z
U)q
TIME IN HOURS
4 6 8TIME IN HOURS
TIME IN HOURS
4 6 8 10 12TIME IN HOURS
FIGS. 2-5. Percent distribution of 14C in fractions recovered at various times after supplying aspartate-U-14C(fig 2), glutamate-U-14C (fig 3), y-aminobutyrate-U-14C (fig 4), and alanine-U-14C (fig 5) to excised, intactenidosperms.
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
PLANT PHYSIOLOGY
TIME IN HOURS TIME IN HOURS
too LEUCIONE - U - 14C 10 C 9 GLUTAMINE-U-*Coa Amino Acids a Amino AcidsovrnicAcids Ogn A
o w
70~~~~~~~~
4 60 0 2
a.-~~~~~~~~~~~~~~~~~U0~~~~~~~~~50 0UA.
0 ~~~~~~~~~~~~~~~~~~at
0~~~~~~~~~
2040 0
z
TIME IN HOURtS TIME IN HOURSFIGS. 6-9. Percent distribution of 14C in fract'ions fecovered at various times after supplying glycine-U-14C
(fig 6), serine-3-14C (fig 7), leucine-U-14C (fig 8), and gluttamine-U-14C (fig 9) to excised, intact endosperms.
-392
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STEW ART AND BEEV-ERS-GLUCONEOGENESIS FRO'M AMINO ACIDS
Table III. Comiiparison of Utiniivation of Labeled Amino Acids by E]ldospermi Halvies
Per cent of 14C recovered after 12 hrs inAmino acid added Aminiilo acids CO, Organic acids Sugars
8 % of the 14C recovered after 12 hours was Dresentin the sugar fraction. In the experiments witharginine, valine, and phenylalanine, the only radio-active amino acid recovered was the one originallyprovided.
To sunmiiiarize the results and to emphasize thecontrastin,g metajbolism of the amino acids providedto the endosperm. the distribution of '-C in variousfractions, after 12 hours is showin in taible III. The
w:w
0
Uj
I--
0I-
0
zz
TIME IN HOURS
FIG. 10. Percent distribution of 14C in fractionsrecovered at various times after supplying proline-U-14C,arginine-U-11C. valine-U-14C, and phenylalanine-U-14C separately to excised, intact endosperms.
main point of emiiphasis is that alanine, aspartate,glycine, serine, and glutamate are utilized extensivelyfor suggar synthesis by the endosperm. Leucine,glutamine, and proline are utilized to a lesser extentand arginine, phenylalanine, and valine virtually notat all. From these results and from the distributionof amino acids in the exudate (table I, column I),it would appear that carbon from the gluconeogenicam,no acids is available for transport to the embryoas sugar whereas the other amino acids are conservedand transported intact.
Translocatory Products fromtl Labeled AmtinoAcids. It is clear from the above results that onlysome of the amino acids present in the protein hy-drolysate of the endosperm undergo appreciable fur-ther metabolic conversion in this tissue. Those whichare rapidly meta(bolized give rise to sucrose as a majormetabolic product and relatively small amounts ofI4CO. are released from the endosperm halves.
Since sucrose, the product of fat utilization is thedominant sugar in the translocation stream as showvnby exudate analvses (9), it wvas clearly of interest toknow if the ability of the endosperm tissue to convertsome amino acids to sugars was reflected in thetranslocatory prodtucts. Four amino acids were con-pared; 3 of these, aspartate, glutamiate, and alaninewere good sugar precursors while valine was not.The labeled amino acids were applied directly to theouter surface of the endosperm of intact seedlingswhich were then maintaiue(i in a moist atmosphereovernight. The hypocotvis w-ere then excised and
Tal)le IN. Raidioactiv'iti ini Various FIraetiomis Deirived froml Hypocotyl Exudate 2-4 lhouris After Application ofLabeled Aminiio Acid to Outer Endosperm}z of Intact Secdlintg
Radioactivity in various fractions (cpm)Total 14C in exudate 26,000 19,600 54,800 26,200'IC in sugars. 21,500 14,000 50,500 800'4C in organic acidIs 700 700 800 4004C in amino acids 3800 4900 3000 25,000including glutaimiine
14C in glutamine 2800 4000 2000 None14C in amino acid added 400 500 100 24,000
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PLANT PHYSIOLOGY
exuidate collected as before. Firactioinationi and 14Canalysis of the exudate provided the results showNn intable IV. From valine, 95 % of the 14C recoveredin the exudate was in the aimino, aci(l fraction and allof this w-as found to be in valine. 'T'hlus the inactivityof this amino acid as a metabolite in the experiments
vitlh excised endospermii halves hold.> also for intactsee(lliIlgs; it is translocated intact into the embry-onicaxis.
By contrast, very large proportions of the 14C inthe exudates from the saimiples receivinig the otheraminio acids were ipresent as sugars (table IV). Therecovery of 1+C in the sugar fractioni exceeded theextent of conversion observed in the exiperiments withexcised endosperm lhalxes and suggested not only thatsucrose was the major fornm in xvhich the C-skeletonsderived from these acids xvere translocated, but alsothat otlher metabolites wxere discrimiiinated against. Itxvas anticipate(d that the "C in the amino acid frac-tion would be preseint in the aminio acid provided, asit was for v\aline. However, from aspartate, gluta-mate, and alanine the compound in the amino acid
fr actioin containing most 14(j ix-as glutamine. Vir-tually Ino 14C wx-as recorered in asparagine and verylittle in the labeled amino acid ad(led to the endo-spleril.
Discussion
The iniportance of glLitamlille as a metabolite(luring the germination of proteinaceous seeds has, ofcouirse, been known for many years (6). The presentxvork establishes that glutamiline is indeed the pre-
dlonlinant form in wNhich nitrogen is transported to theseedling axis in the castor bean seedling. However,the most ;ignificant and novel aspect of the resultsis the demonstration that in this particular seedlingcarbon froml aspartate, glutamate, alanine, glycine.and serine is actively coinverted to sticrose in the(non-ch.lorophyllous) endosperm aicl is theni trans-nitted to the seedl'ing axis.
Novw, although the possibi lity of gluconeogenesisfrom amlino acids in germinating seedlings was con-sidered inl the classical xvork (6) it lhas never to outrknioled-ge been experimentally tlemonstrated, andI inprevious xvork with pea seedlings using the present
metlhods no suchi conversion was ol)serxved (11). Itappears that the production of stigars from particularamino acids in the castor bleall endosperiii is due tothe fact that these amino acids become available fronibreakdown of protein at the sanie timiie as a massive
production of sucrose from fat is taking place. Largescale gluconeogenesis fromi particular amino acids may
wxell be confined to suclh fattx seedlings.In this ipaper we have beeni concernied more xvitl
the fact that gluconeogenesis occur-s from alaniie.serine, glycine, aspartic and glutarnic acids than Awiththe pathways of conversioni. AMost oIf the conversionlscan in fact be accounted for bx- invoking reactionsdemon.strated previously in plant niaterials (8, 17)
wvhiclh lead to interme(diates in the conversion of tatto sucrose. lraiisamination of above am,ino acids
x-ould lead to keto-acids which eitlher are part of thatreactioln sequence ( gflyoxylate, oxalacetate) or arereadily converted inlto suclh initerimiedliates (hvdroxx -
py ruvate, a-keto-laitarate, y-amiiinobuityrate, pyrnl-vate). The productilon of labeled organic acids whiclhwas obserxed, alu(i particularly the temiiporary accumu-lationl oft'' -C' maate from caan be accounted
for oni this l)asi;.The deiiiont-trated iproductioni of glutainate-_4C
from proline-'"'C, wxxhich has been observed previouslyill other tisiies ( 18, 21) prolbably accounts for theminor con-xver:<:on of prolinie carboln to sucrose and tog,lutalminie, ancl the ability of letucine to provide acetyluin7ts (22 ) mav' explain tihe suicrose prodtlction fromthis amilno acid.
The aminio a.cid composition of the exu(late (table1) and the re.ults of the experimiienits hovowing the4-C prodtucts present in tlle extidate after apply inglabeled amino acidls (table IV) emiiphasize the impor-tance of the above reactions in intact seedlings.Glutanmiate, glvcinc , aspartate. alanine. and serine, theamino acids ictivelv converted to sucrose in the endo-spermii halv-es ( table III) eaclh contribute mlluch less
to the amiiino acid comiiposition of the extudate than tothe proteiin iromii Nxvhiclh they are dleriv ed; glutamiate,alanine, anI aspartate are l)re-eilt in paarticularly smlalltmounts ( tah'e I ). \Wleni these labele(d amnino acidiswvere appliedl to the outside of intact enidospermiistabhe IN,") most of the toC appearing in the exuidate
froml the hypIbocot1l vas in sucrose and( x-irtually nonexacs prelent is tihe aimino acid ad(led. By contra-st,valine, which was mi etabolized extremely slowly wlhenadded to excised endospermli halves (fig 10) and didnot give riSe to appreciable aamounts of suicrose, wasfound ulichialngedl in tile hlpocotvl exudate when itvas ad(de(d to intact endosperiml.
\Ne conclude thien that in the intact scdlin'g thoseamiinio acids xx--lich have )een s,hown to be convertibleto sucrose are so converted and that it is largelv illthis forimi that the&r carbon i ililoxed to the embryoproper. TI>he loxx amounts of these amiiino acids in
the trai1-1ocat:oil stream mu,st l)e accouniited for hvtheir active miietaholism in the endlosperm, since wehave shown that when a variety of amiiino acids xvassuppliedI to cotyledons of excised seedlings in thesystemi used Il kriedemann antd Beevers (9) all x-ereabsorlbedl there x\-as no dislcrimination against thle,Iticoneog,enic a1ilnO acids. T-hose amino acids suchlas valine, xvhich (ire not gluconeogenic, are apparentlydirectl trasferre(d tlhrougl tile cotvledons into tiletranslocation streaimi.
Ini adlditiOnl to such amiiino acids, anl excecdingthem greatly in quantity in thle translocatioin streamis gititamiline. wxxhich accouints- for 40 % of the nitro-elolis coillpolllnd; ( table I). It seemis reason--able toconclude that the anmide iiitrogen is (lerived largelyfrom the dealllinatioln of tile -gluconieogenic amllinoacidcs. Hoxxever, it is also clear that some ot tllecarbon fromi these aiiino aci(ls is a>-o trail-ocated
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STEWN'ART AND BEEV'ERS-GLUCONEOGENESIS FROM AMINO ACIDS
as glutamine. This suggests that newly formed glu-tamate, in addition to that produced from hydrolysisof reserve protein, contributes to glutamine formation.It is of interest that, in contrast to glutamine, aspar-gine does not appear to function as a transporter ofNH., groups in the castor bean seedl,n,g.
Literature Cited
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4. BENEDICT, C. R. AND H. BEEVERS. 1961. Forma-tionl of sucrose from malate in germinatiing castorbeans. I. Conversion of malate to phosphoenol-pyruvate. Plant Physiol. 36: 540-44.
5. CANVIN, D. T. AND H. BEEVERS. 1961. Sucrosesynthesis from acetate in the germiniating castorbean: kinetics and pathvay. J. Biol. Chem. 236:98-95.
6. CHIBNALI -A. C. 1939. Proteini metabolism in theplant. Yale University Press, Ne\v Haveni. Con-necticut.
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York.9. KRIEDEIMANN, P. AND H. BEEVERS. 1967. Sugar
uptake and translocationi in the castor bean seed-lings. I. Characteristics of transfer in intact andexcised seedlings. Plant Physiol. 42: 161-73.
10. KRIEDE-MANN. P. AND H. BEEV-ERS. 1967. Sugaruptake and translocation in the castor beani seed-ling. II. Sugar transformations during uptake.Plant Physiol. 42: 174-80.
11. LARSON. L. A. AND H. BEEVERS. 1965. Amiinioacid metabolism in young pea seedlings. PlantPhysiol. 40: 42432.
12. MOORE. S. AN-D W. H. STEIN. 1954. A mi1odifiedninhydrin reagelnt for the photometric determina-tionl of amino acids and related compound-s. J.Biol. Clheml. 211: 907-13.
13. MORRIS, C. J. AND J. F. THOMPSON. 1965. CoIn--ersion of ni-carboxy-phenyialanine to in-carboxyphlenvlglycinie in WVedgewood iris leaves. Arch.Biocheml. Biophys. 110: 506-10.
14. NEAL. G. E. AND H. BEEVERS. 1960. Pvruvateuitilization in castor bean enidospermii and (othertissues. Bioclheml. J. 74: 409-16.
15. OAKS, A. 1965. The regulation of nitrogen lossfrom maize endosperm. Can. J. Bo:any 43: 1077-82.
16. OAKs, A. 1966. Tranisport of amino acids to tilemaize root. Planit Phvsiol. 41: 173-80.
17. STEWN-ARD, F. C. AND D. J. DURZAN-. 1964. \e-tabolism of niitrogenous compounds. In: PlanitPhysiology. A Treatise. F. C. Steward. ed.Chapter 4. V"ol. IV A, Academiic Press. NewvYork.
18. STEWN-ART, C. R. 1967. The effects of water andoxygeni content on changes in aminio acid contentof excised leaves and studies on proline metab-olism in wilted anid unwilted leaves. Ph.D. Thesis,Cornell University, Ithaca, New- York.
19. STILLER, M. L., G. E. NEAL, AND H. BEEVERS.1958. CO., fi.xation during the conversion otfat to carblohydrate in the castor bean. PlantPhv-siol. 33: xxxiv.
20. THOMPSON. J. F. AND C. J. MORRIS. 1959. Thiedeterminationi of amino acids from plantspaper chromiiatography. Anal. Clhem. 31: 1031-37.
21. Tupv-. J. 1964. 'Metabolism of proline in stylesand pollen tubes of \Xicotiana ala7tai L. In: PollenPhysiology andI Fertilization. H. F. Liniskens,e(l. North Holland Publishing Company. Anm-sterdam. p 86-94.
22. WHITE. A.. P. HANDLER, AND E. L. SMITH. 1964.Principles of Biochemistry. Chapter 27. 'McGraw-Hill Book Company, Ncw York.
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