changes in the levels of major sulfur metabolites amino ... · peas (pisum sativum l.) line pi/g...

Plant Physiol. (1987) 83, 354-3590032-0889/87/83/0354/06/$0 1.00/0

Changes in the Levels of Major Sulfur Metabolites and FreeAmino Acids in Pea Cotyledons Recovering from SulfurDeficiency

Received for publication February 21, 1986 and in revised form October 14, 1986

PETER K. MACNICOL* AND PETER J. RANDALLDivision ofPlant Industry, Commonwealth Scientific and Industrial Research Organization,Canberra, A.C. T. 2601, Australia

ABSTRACT

Changes in levels of sulfur metabolites and free amino acids werefollowed in cotyledons of sulfur-deficient, developing pea seeds (Pisumsativum L.) for 24 hours after resupply of sulfate, during which time thelegumin mRNA levels returned almost to normal. Two recovery situationswere studied: cultured seeds, with sulfate added to the medium, and seedsattached to the intact plant, with sulfate added to the roots. In bothsituations the levels of cysteine, glutathione, and methionine rose rapidly,glutathione exhibiting an initial lag. In attached but not cultured seedsmethionine markedly overshot the level normally found in sulfur-suffi-cient seeds. In the cultured seed S-adenosylmethionine (AdoMet), butnot S-methylmethionine, showed a sustained rise; in the attached seedthe changes were slight. The composition of the free amino acid pool didnot change substantially in either recovery situation. In the cultured seedthe large rise in AdoMet level occurred equally in nonrecovering seeds.It was accompanied by 6-fold and 10-fold increases in 'y-aminobutyrateand alanine, respectively. These effects are attributed to wounding re-sulting from excision of the seed. 3S-labeling experiments showed thatthere was no significant accumulation of label in unidentified sulfur-containing amino compounds in either recovery situation. It was concludedfrom these results and those of other workers that, at the present levelof knowledge, the most probable candidate for a 'signal' compound,eliciting recovery of legumin mRNA level in response to sulfur-feeding,is cysteine.

Apart from its marked effects on plant appearance and yield(19), S deficiency induces a rather consistent syndrome of bio-chemical changes. Conspicuous among these is a greatly ex-panded free amino acid pool with a distorted composition rela-tive to nondeficient plants, and depressed levels of S amino acids.This altered composition is caused by a large accumulation ofparticular amino acids such as arginine, asparagine, and glycine,and is seen in leaves (4, 21, 22), roots (22), and seeds (12) ofplants in various families. Striking changes are also seen in theproteins of S-deficient plants, especially in the storage proteinsof the seed, but also to a lesser extent in leaves (16). Thus inmature S-deficient seeds ofboth cereals and legumes a few majorproteins of higher than average S amino acid content are absentor nearly so (8). Recent work with peas (Pisum sativum L.) hasshown that the nonaccumulation of legumin and pea albumin 1in developing S-deficient cotyledons is due to cessation of theirsynthesis caused in turn by very low levels of their mRNAs (3).However, the transcription of legumin messenger in a cell-freesystem is little affected by S deficiency (1); the mechanism of

control of mRNA levels is in fact- not understood.During the recovery of seeds from S deficiency, initiated by

the feeding of intact pea plants or isolated seeds with inorganicsulfate, the synthesis of these proteins and their mRNA levelsare restored to normal within 24 h (3). During such recovery thebehavior of the levels of the major S metabolites and the timecourse of these changes are of considerable interest. Irrespectiveof the exact mechanism of control of mRNA levels, it is aplausible assumption that the changed S status of the tissue issignaled by the changed level of some low mol wt S compound.Data on the extent and kinetics of replenishment of the pools ofthese metabolites may therefore reveal such a 'signal' molecule.Such information, together with measurement of the non-S freeamino acids, will also show whether the biochemical syndromeof S deficiency is reversible in terms of metabolite levels, overthe time scale ofmRNA recovery.

MATERIALS AND METHODS

Plant Material. Peas (Pisum sativum L.) line PI/G 086, se-lected from cv Greenfeast, were grown in artifically lit cabinetssupplying 300 to 400 gE m-2-s' for a 16 h photoperiod. Seed-lings were raised in trays of vermiculite and 14 d after sowingwere transplanted into 15 cm pots. Sulfur-sufficient plants weregrown in perlite/vermiculite (1/1, v/v) with complete nutrientsolution. The conditions were those described by Millerd andSpencer (17) except that the temperature was 20C. S-deficientplants were grown under similar conditions in sand/perlite (1/1,v/v). The nutrient solution of Randall et al. (18) was used. Thesulfate concentration was kept at 0.25 mm for 2 weeks aftertransplanting and then lowered to 0.025 mm until the first flowersopened, approximately 5 weeks after transplanting, when nu-trient solution without sulfate was given. Sulfate was supplied asMgSO4 which was replaced as required by MgCl2 in order tomaintain Mg constant. S-deficient plants showed characteristicsymptoms and were smaller with fewer pods. Selected pods were19 d after flowering when sampled at the beginning of therecovery periods in the various experiments, and for uniformityonly those from nodes 3 to 6 were used.Recovery ofwhole plants was initiated in a group of S-deficient

plants, selected for uniformity, by flushing the pots with nutrientsolution containing 2 mm sulfate. At specified times thereafter apod was harvested, and the cotyledons immediately dissectedfrom the seeds and dropped into liquid N2, where they werestored prior to analysis. The next day this procedure was repeatedwith other plants from the same batch, thus providing a replicateset of samples. Seeds from the recovering plants are designated-S+S to distinguish them from seeds from S-deficient untreatedplants (-S-S) and S-sufficient plants (+S+S).

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SULFUR METABOLITES IN PEA COTYLEDONS

Seed Culture. Seeds were excised and placed with the cutfunicle in 250 ,l of the appropriate nutrient solution in a smallplastic vial-cap. They were exposed to a light intensity of about35 uE .m-2 s-' in humidified glass dishes on the laboratorybench. The basal ('-S') solution was 4 mM CaCl2, 4 mM KCl, 4mM MgCl2, 6 mM KH2PO4, 114 mM L-asparagine, 39 mM L-glutamine, 292 mm sucrose, plus micronutrients as in Randall etal. (18). For 'recovering' seeds this medium was supplementedwith 6 mm K2SO4 + 6 mM MgSO4, except in the case of seedsthat were fed [35S]sulfate (see below), where recovery was initiatedwith 12 mm glutathione. Each treatment consisted of 12 seeds,randomized with respect both to plant and position within thepod. At the completion of the experimental period the seeds ina given treatment were put on ice. The cotyledons were quicklydissected out and distributed into three samples for analysis,using a permutative scheme; these were weighed and stored inliquid N2. S-deficient seeds cultured without S and recoveringseeds are referred to as -S-S and -S+S, respectively.

Labeling of Seeds with V35S]Sulfate. In order to label culturedseeds, on either S-containing or S-free medium, 70 ,uCi of [35S]-sulfate were added to the medium bathing each seed. In the caseof seeds attached to intact plants, 150 uCi in 30 gl were injectedinto the pod pedicel.Assay of Legumin mRNA. Cotyledons were powdered under

liquid N2 and total RNA was extracted as described by Chandleret al. (2). The procedure includes precipitation with 2 M lithiumacetate, resulting in a product which is free of DNA and largelyfree of 4S and 5S RNA. Hybridizations to determine the levelsof legumin mRNA were done essentially according to White andBancroft (24) using the legumin cDNA plasmid pPS 15-75 (2).

Chemicals. L-[U-`4C]Methionine, [35S]Na2SO4 (carrier-free,aqueous solution, pH 6-8), [3H]dansyl chloride, and ['4C]dansylchloride were purchased from Amersham (Bucks., UK) and L-[U-_4C]cysteine and L-[glycine-2-3H]glutathione from New Eng-land Nuclear. Unlabeled dansyl chloride, dansyl-methionine, andglutathione were from Sigma. Markers of dansyl-S-carboxyme-thylcysteine and dansyl-S-carboxymethylglutathione were pre-pared as in Macnicol and Bergmann (15). F1700 polyamide foils-were obtained from Schleicher and Schull (Dassel, FRG).

Metabolite Extraction. Cotyledons were powdered under liq-uid N2 and homogenized in cold 10% TCA as previously de-scribed ( 13). A 100 yl aliquot (equivalent to 20 mg of tissue) wasanalyzed immediately for GSH as described below; gradual hy-drolysis of the y-glutamyl bond occurs on storage in TCA evenat -20°C (PK Macnicol, unpublished data). Further 100 ,laliquots for analysis of Cys and Met as well as non-S aminoacids, and a 400 ,d aliquot for MeMet' and AdoMet, were storedat -200C.

Glutathione Analysis. A 100 ,d homogenate aliquot was spikedin an ice bath with 5 gl of 18 tLM GSH containing 500 nCi of[3H] GSH, then centrifuged at 20,000g for 5 min and thesupernatant carefully transferred to another tube, where the TCAwas quickly removed by three extractions each with 0.5 ml ofcold, water-saturated diethyl ether. The residual aqueous phasewas rotary-evaporated to dryness at 40°C and the residue dis-solved in 100 ju of 50 mm Li2CO3 buffer, pH 9.5. The GSH wasreduced and carboxymethylated with 1 ,ul of 200 mM DTT (letstand 30 min at room temperature), followed by 1 ul of 1 Miodoacetic acid in 1 M NaHCO3 (60 min at room temperature),followed by 1 A1 of 800 mm mercaptoethanol. A 10 ,u aliquot of

Abbreviations: MeMet, S-methylmethionine; oyAbu, y-aminobutyr-ate; AdoMet, S-adenosylmethionine; CMC, S-carboxymethylcysteine;CMG, S-carboxymethylglutathione; DNS, dansyl = 5-dimethylaminon-aphthalene-l-sulfonyl; GSH, glutathione; Hcy, homocysteine;Met(O),methionine sulfoxide. Abbreviations for protein amino acids arestandard International Union of Biochemistry nomenclature.

this solution was reacted at 25°C for 30 min with 5 Al of 5 mmdansyl chloride in acetonitrile, containing 1 ACi of ['4C]dansylchloride. After stopping the reaction with 1 Al of 2% (w/v)diethylamine hydrochloride, 4 AI of reaction mixture were spot-ted onto a polyamide thin layer together with carrier dansyl-S-carboxymethylglutathione (DNS-CMG) and the chromatogramdeveloped once in 2% HCOOH (first direction) followed by 4times in chloroform/2-butanone/acetic acid (8/1/1, v/v/v) (sec-ond direction). The DNS-CMG spot was eluted and counted asin Macnicol (12); the '4C/3H ratio was linearly proportional toamount ofGSH over the range 0.05 to 5 nmol GSH.

Analysis of Other S-Metabolites and Free Amino Acids. Cysand Met were determined separately from the other amino acidsbecause their low initial levels necessitated using [3H]dansylchloride of higher specific activity. A 100 Al aliquot of homoge-nate was spiked with 33 nCi of [14C]cystine and 33 nCi of [14C]-Met in 5 Al of 1 mM DTT, and the steps of ether extraction,reduction, carboxymethylation, and dansylation were carried outas for GSH, except that 12.5 ACi of 5 mm [3H]dansyl chloridewas used. After adding carrier DNS-Met to the origin of thepolyamide thin layer, it was partially oxidized by overspottingwith 0.6% (w/v) H202 and immediately drying again, in orderto provide carrier DNS-Met(O). Carrier DNS-CMC was thenspotted, followed by 3 Al of reaction mixture. The chromatogramwas developed once with 2% HCOOH (first direction) and twicewith benzene/acetic acid (9/1, v/v) (second direction). Aftercutting out the spots of DNS-Met and DNS-Met(O), the chro-matogram was stapled together and redeveloped twice in thesecond direction with benzene/acetic acid (3/1l,v/v) to resolveDNS-CMC. Elution and counting were as in Macnicol (13); thecounts in DNS-Met and DNS-Met(O) were summed. The pres-ence of DNS-Met(O) was assumed to result from autoxidationof Met during extraction and chromatography. It was foundnecessary to cut out and elute blank spots adjacent to the threeDNS-amino acid spots, in order to correct for nonspecific smear-ing of low levels of 3H across the chromatogram.MeMet and AdoMet were determined in a 400 Al aliquot of

homogenate as in Macnicol (14). In this method quantitation isvia [3H]dansyl-[14C]homoserine, after spiking with '4C-labeledMeMet and AdoMet, separation on a phosphocellulose columnand heat treatment.

Non-S-free amino acids were analyzed in a 100 Al homogenatealiquot as previously described (13).

RESULTS

Sampling, Analytical Precision, and Statistical Treatment ofData. For both types of recovery experiment (see below), thepoints shown for S metabolite levels are the means of analysesoftwo replicate samples of 8 to 24 cotyledons, except for MeMetwhere only one replicate was analyzed. Analytical precision wasgenerally within 10%.

Standard errors of the differences between means were calcu-lated for all S metabolites except MeMet. It was necessary totransform the data to logarithms before analysis of variance tostabilize the variation. Significant treatment effects are shown inTable I, which can be referred to when interpreting Figures 1and 2.

Recovery in Cultured Seeds. When S-deficient seeds werecultured on nutrient medium supplemented with 12 mM inor-ganic sulfate ('-S+S'), the dot-blot assays showed a markedincrease in the level of legumin mRNA in the cotyledons overthe first 12 h, increasing further during the next 12 h. The dataare not shown because this agrees with previous work (3); by 48h the mRNA level was close to that in S-sufficient cotyledons.

Figure 1 presents the changes that occurred during this first 24h of recovery in the levels of five prominent S metabolites. Thefree amino acids Cys and Met increased right from the start, and

355

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MACNICOL AND RANDALL

Cultured seed

Plant Physiol. Vol. 83, 1987

Intact plant400 - GSH 0

200

O*-0-_ S-S

-S+S 0

C-0.

0

4-

Ec

s____ ~~~_ s s 0

C

0

-0a1)

-5

EcF~~~~~~ - ----- - ----

20)

25 ME MET

--

200 ADO MET -S

100 _ _

0 6 12 18 24

Hours

FIG. 1. Changes in levels of sulfur metabolites in cotyledons of re-

covering (-S+S) and nonrecovering (-S-S) S-deficient, cultured peaseeds.

at 24 h had reached levels close to those in S-sufficient cotyledons(about 100 and 45 nmol/cotyledon pair,2 respectively); Metshowed a much smaller proportional increase than Cys. The Cys-tripeptide GSH rose only after a lag of 2 to 4 h, and reached alevel well below the S-sufficient value (about 500 nmol/cotyledonpair2). The sulfonium compounds AdoMet and MeMet showedcontrasting behavior to Cys, Met and GSH, in that the levelincreased almost equally in recovering and nonrecovering coty-ledons. While AdoMet rose strongly and reached a level severaltimes above that in S-sufficient cotyledons (about 60 nmol/cotyledon pair2), MeMet increased only slightly and remainedwell under the S-sufficient value (about 25 nmol/cotyledonpair2). Since, as discussed below, this behavior ofAdoMet in thenonrecovering situation suggested that the cotyledons were re-acting to excision per se, the S-metabolite levels were also fol-lowed in attached cotyledons.

Recovery in the Intact Plant. Supply of 2 mm sulfate to theroots of S-deficient plants elicited recovery of legumin mRNAlevel in the cotyledons at approximately the same rate as incultured cotyledons (data not shown), as previously demon-strated (3). Figure 2 gives the changes in S-metabolite levelsduring the first 24 h of this recovery. Cys, Met, and GSHexhibited changes quite similar to those in the cultured cotyle-dons, and again there was a 2 to 4 h lag in the accumulation of

2 These levels were measured in S-sufficient seeds of the same age ( 19d after flowering) as S-deficient seeds. The values for Cys and Met are

means for three separate experiments, those for GSH, AdoMet, andMeMet are for one experiment.

-s -s

0

140-METO-5+5

70 -

0-25 ME MET

F_-0~~~~~100 - ADO MET

,0- _o s+ S

O ..I

0 6 12 18 24

Hours

FIG. 2. Changes in levels of sulfur metabolites in cotyledons of re-

covering (-S+S) and nonrecovering (-S-S) S-deficient, attached pea

seeds (whole plant recovery). Note changed ordinate scales relative toFigure 1.

GSH. However, with Met and GSH the extent of the increasewas far greater than in the cultured seed (note the differingordinate scales in Figs. and 2), particularly during the second12 h of recovery; the level ofMet reached was much greater thanthat in S-sufficient cotyledons. The changes in levels of thesulfonium compounds were relatively small, both rising onlyduring the first 4 h of recovery. This behavior of AdoMetcontrasts particularly with that in the cultured seed.Changes in Non-Sulfur Amino Acids. Figure 3 gives the com-

position of the free amino acid pool in recovering (-S+S) andnonrecovering (-S-S) cotyledons from cultured seeds and intactplants, and in S-sufficient (+S+S) cotyledons from intact plantsfor comparison. Not only is the accumulation of arginine, aspar-agine, and some other amino acids in S-deficient cotyledonsclearly seen, but it is also evident that in neither recoverysituation was there any substantial reversion to the controlcomposition during the 24 h treatment period.

It was noticeable that the changes due to culturing seeds werein several cases greater than any changes due to recovery. Thusin cultured but not attached seeds there were striking increasesin the level of yAbu (6-fold after 24 h) and Ala (10-fold); severalother amino acids (Asn, Glu, Leu, Lys, Phe, Pro, Tyr) increased2- to 3-fold (zero-time data not shown).

35S-Labeling Experiments. These were undertaken in order tolearn whether there is an accumulation, during recovery, of anyS compound(s) not covered in the above analyses. After 4 and12 h, the cotyledons were extracted as in "Materials and Meth-ods" and the extract was reacted with nonradioactive dansylchloride. After purification of the dansyl fraction (11) it was

chromatographed on polyamide layers in the two-dimensionalsystem of Macnicol and Bergmann (15) and that of Laatsch (9),

356

( L-1-

2

1

I Il




50r

40

C.)

01)25

E

25h

50r

2)

a)

E

25

0--

Cultured seed-S+S,24H-S -S. 24H

5ozoWZD-WWW W ZcrWrx>-w

Intact plant

--S-S. 24H-S-S, 24 H

D50<Oz Wz D > OzUlW Den F u Wo z Lz j

co

0 Normala)"O 20-0)

100

-

FIG. 3. Composition of free amino acid pool ofcotyledons of recover-ing (-S+S) and nonrecovering (-S-S) S-deficient pea seeds after 24 h,recovery occurring either in the cultured seed or the intact plant. Freeamino acid pool of normal (S-sufficient) cotyledons shown for compar-ison.

who published a spot-map for over 90 amino compounds. Thelabeled spots were detected by fluorography.

In order to be able to feed [35S]sulfate to cultured seeds andnot incur a large dilution of specific activity, recovery wasinitiated with GSH instead of sulfate (3). In 24 h there wassubstantial recovery of legumin mRNA level, in agreement withprevious findings (3). After 3 weeks of fluorography only threeradioactive spots were found on the chromatograms, two ofwhich were identified as CMC and CMG (cf. Ref. 15). The thirdradioactive spot did not coincide with any fluorescent spot orwith any spot on the Laatsch map. The order of relative intensitywas CMC > third spot > CMG both at 4 and 12 h. No labelingwas seen in Met or Met(O). The third spot was subsequentlyidentified by co-chromatography in several solvent systems asDNS-S-carboxymethylhomocysteine The labeling of the actualmetabolites was therefore Cys > Hcy > GSH.When recovering seeds on the intact plant were fed [35S]sulfate

by injection into the pod pedicel, label was found in the abovethree metabolites, but also in Met and Met(O). In this casefluorography was for 7 weeks. The order of intensity both at 4and 12 h was GSH>> Cys > Met, Met(O) > Hcy. Weak labelingwas also seen in a spot moving in the correct position (9) for bis-DNS-cystathionine.

DISCUSSION

The selective effects of S nutrition on the accumulation ofseveral major seed proteins make the pea an attractive systemfor studying the regulation of storage protein composition. Al-though it has been demonstrated that the rate of synthesis ofthese proteins correlates closely with their respective mRNAlevels (3), there are in turn several possible mechanisms ofcontrolof mRNA abundance in response to S status. The results of

Beach et al. (1) for legumin make transcriptional control unlikely.Two possibilities for control at the post-transcriptional level areselective inhibition of messenger processing and preferentialdegradation of messenger due to inhibited translation (3). Ineither case, it seems reasonable to postulate that the improved Sstatus of the cotyledon is signaled by the increasing concentrationof a S-containing compound.The S compounds shown to initiate recovery of legumin

mRNA level in S-deficient pea seeds are inorganic sulfate, Cys,Met, GSH, and mercaptoethanol (3). This indicates that the'signal' molecule is probably a reduced S compound, unless oneassumes that each of the above compounds is first broken downto sulfate. In the case of Met this is unlikely, as Datko and Mudd(6) could not detect formation of [33S]sulfate from exogenousI35S]Met in Lemna. Obvious criteria for such a signal compoundare a marked rise in level, confined to recovering cotyledons,and showing appropriate kinetics, i.e. the rise has to occur beforemessenger recovery is detectable. KWith these considerations in mind, the changes in levels of

five majorS-metabolites were determined in pea cotyledons inwhich recovery of legumin mRNA was occurring (first becom-ming detectable at 12 h) in either of two situations: in thecultured, isolated seed or in the seed attached to the intact plant.In both situations the behavior of Cys, Met, and GSH wasessentially the same: a marked rise which only in the case ofGSH was preceded by a lag of several hours. On the other hand,the behavior of AdoMet was clearly different between the tworecovering systems, the strong rise in the cultured seed occurringindependently of recovery. The trends shown by MeMet werehardly significant. For convenience, the proportional extent ofthe changes in level of the five metabolites is shown in Table I,together with estimates of statistical significance.On the basis of the criteria set out above for a signal compound,

AdoMet and MeMet may be eliminated from consideration. Theinitial lag shown by GSH, together with a likely Cys-storagefunction, reduce the probability of such a role for this metabolite.The comparison of Cys and Met clearly favors Cys, as it showsmuch larger relative changes than Met, particularly at early times.The rise in Met level was never more than 2-fold within 12 h. (Itis of interest that in S-sufficient seeds also, Cys levels vary morethan Met. In different experiments over a 2 year period Cyslevels in S-sufficient seeds ranged from 50 to 150 nmol/cotyledonpair. The corresponding range for Met was 40 to 50 nmol/cotyledon pair [PK Macnicol, unpublished data].) Although theCys level varies between wider limits than Met it may be suffi-ciently tightly controlled within a given physiological situation

Table I. Change in S-Metabolite Level in Recovering CotyledonsRelative to Nonrecovering Cotyledons

Calculated from data of Figures I and 2.

Ratio.Level in -S+SRecovery Metabolite Level in -S-SSituation Mtblt

4h 12h 24h

Cultured seed Cys 1.5 4.7** 8.4**Met 1.2 1.1 1.3GSH 1.6 2.8** 6.1***AdoMet 1.0 1.0 0.9MeMet 0.7 1.1 0.9

Intact plant Cys 3.4** 7.1 9.0***Met 1.6 1.9 5.0**GSH 4.8* 16*** 50***AdoMet 2.4 1.3 1.2MeMet 1.4 1.0 1.4

Values followed by asterisks are significantly different from 1.0;>,

Ul-I -- -- - 1..

O .I

357



MACNICOL AND RANDALL

for a severalfold increase, such as seen here, to act as a signal.Because of the compartmentation of Cys in plant tissues into asmaller active pool and a larger inactive pool (7), a much largerproportional increase may be occurring in the active pool thanis seen in measurements of the total pool.

Further support for a signal role of Cys rather than Met isfound in a mechanism that can be postulated for the control ofmRNA levels. Sequencing ofcDNA clones (P Chandler, unpub-lished data) has revealed an interesting difference in cysteinecodon usage between legumin and pea albumin 1 on the onehand and vicilin on the other, in the pea. Only TGT is used bymRNAs for three major vicilin Mr 50,000 polypeptides, whereasmRNAs for both legumin and pea albumin (PA- 1) utilize bothTGT and TG codons. The synthesis of vicilin is not reduced byS deficiency (3). The failure of legumin or PA-1 mRNAs toaccumulate in S-deficient seeds could therefore be due to lack ofthe tRNACy) isoacceptor bearing the appropriate anticodon, re-sulting in translational stalling and subsequent mRNA break-down. The most likely signal for resynthesis of this isoacceptorwould be the free Cys level. This hypothesis is currently beingtested.

In spite of these arguments for Cys, some evidence can beassembled favoring Met as a signal. First, in a recent study of theinhibition of formation of the f#-subunit of 7S storage protein(conglycinin) in cultured soybean cotyledons, Creason et al. (5)concluded that Met and its analog S-ethylcysteine act directly as'effectors' to prevent the formation of the (-subunit mRNA,even though the increase in tissue Met was only about 3-fold. Inthese experiments, however, mRNA levels were not assayed, andeffects on the legumin-like I I S protein (glycinin) were not shown.Second, while it is very probable that the sulfur atom of thecompounds found to initiate legumin mRNA recovery (3) canbe recycled into Met, the conversion of Met to Cys (reversetranssulfuration) is scarcely detectable in most plants (6). Never-theless, it is in legumes themselves that evidence for this conver-sion has been found (6). Third, since the level of free Met in peacotyledons is more tightly controlled than that of Cys, at leastwith respect to S-status (12), a relatively small perturbation ofMet level may suffice as a signal. However, Met has one obviousdisadvantage as a signal in the present context: the fact that it isrequired for the initiation of synthesis of all proteins.The striking increases in levels of AdoMet, yAbu, and Ala in

the cultured seed may well be a wounding response. This sugges-tion is supported by recent experiments (23) in which the levelsof SyAbu and Ala, but not Glu and Gly, rose spectacularly insoybean leaflets as a result of excision, rolling, or crushing. Therising AdoMet level may reflect an accumulation of ATP, re-quired for its synthesis, such as occurs in leaf discs within hoursof excision (10).A conspicuous difference between the two recovery situations

was seen in the ultimate levels ofGSH and Met that were reached.Not only were they far greater in the intact plant, but the Metlevel overshot the normal S-sufficient value 4-fold. This suggeststhat the recovering isolated seed lacks factors supplied by theparent plant that are necessary for Met synthesis. The nature ofthese factors is unclear. Although the aspartate pathway is likelyto be shut down in the S-deficient seed by the accumulated Thr,Lys, and AdoMet (20), there is excess 4-carbon precursor avail-able, if one assumes that it is accessible to the Met-synthesisingcompartment. The IIS-labeling data, while not quantitative,showed label accumulating in Hcy but not Met, suggesting thatflux through the final step of Met synthesis is restricted in thecultured seed.Apart from Cys, Met, GSH, AdoMet and MeMet, two other

potential signal metabolites are Hcy and cystathionine. Not onlyare these compounds present at very low concentration (PKMacnicol, unpublished data), but radioactive 'spikes' of suffi-

ciently high specific activity were not available for their analysis.While the labeling experiments showed that in both recoveringsituations proportionately little 35S had accumulated in thesemetabolites after 4 and 12 h of recovery, for reasons relating tolabeling kinetics and pool sizes this does not preclude increasesin concentration. What the labeling data did show is that therewas no significant accumulation ofany unidentified S-containingcompound bearing an amino acid group (i.e. capable of forminga dansyl derivative), during recovery.The results for non-S amino acids provide no evidence for any

link between levels of these compounds and recovery ofmRNAlevels. Free amino acid levels showed no substantial reversibilityof the S-deficiency syndrome during the 24 h recovery period.The only relevant previous study appears to be that of Steward'sgroup (21), where 5 to 8 d were needed for the accumulation ofarginine in leaves of mint plants recovering from S deficiency inthe light to diminish back to control values.The present results have narrowed the possibilities for a signal

compound by showing that none of the metabolites Cys, Met,GSH, AdoMet, or MeMet, nor any other S compound with aprimary amino group, exhibits a spectacular rise in level, in bothattached and detached cotyledons, within the time needed formessenger recovery. If, notwithstanding, one ofthese metabolitesconveys the signal, the balance of probability favors Cys. In theabsence of a suitable cell-free system, the identification of thephysiological signal may have to await elucidation of the mech-anism of control of legumin mRNA level.

Acknowledgments-We wish to thank P. Chandler for valuable discussions, D.Spencer and E. Newbigin for legumin mRNA assays, J. Cassells, L. Stipnieks, andM. Rowland for technical assistance, and D. McCann for typing.

LITERATURE CITED

1. BEACH LR, D SPENCER, PJ RANDALL, TJV HIGGINS 1985 Transcriptional andpost-transcriptional regulation of storage protein gene expression in sulfur-deficient pea seeds. Nucleic Acids Res 13: 999-1013

2. CHANDLER PM, TJV HIGGINS, PJ RANDALL, D SPENCER 1983 Regulation oflegumin levels in developing pea seeds under conditions of sulfur deficiency.Rates of legumin synthesis and levels of legumin mRNA. Plant Physiol 71:47-54

3. CHANDLER PM, D SPENCER, PJ RANDALL, TJV HIGGINS 1984 Influence ofsulfur nutrition on developmental patterns of some major pea seed proteinsand their mRNAs. Plant Physiol 75: 651-657

4. COLEMAN RG 1957 The effect of sulphur deficiency on the free amino acidsof some plants. Aust J Biol Sci 10: 50-56

5. CREASON GL, JF THOMPSON, JT MADISON 1985 Methionine analogs inhibitproduction of ,8-subunit of soybean 7S protein. Phytochemistry 24: 1147-1150

6. DATKO AH, SH MUDD 1984 Responses of sulfur-containing compounds inLemna paucicostata Hegelm. 6746 to changes in availability ofsulfur sources.Plant Physiol 75: 474-479

7. GIOVANELLI J, SH MUDD, AH DATKO 1980 Sulfur amino acids in plants. InPK Stumpf, EE Conn, eds, The Biochemistry of Plants. A ComprehensiveTreatise, Vol 5. Academic Press, New York, pp 453-505

8. HIGGINs TJV 1984 Synthesis and regulation of major proteins in seeds. AnnuRev Plant Physiol 35: 191-221

9. LAATSCH H 1979 Identifizierung seltener Aminosauren durch Mikrodansyli-erung. J Chromatogr 173: 398-402

10. MACNICOL PK 1976 Rapid metabolic changes in the wounding response ofleaf discs following excision. Plant Physiol 57: 80-84

1 1. MACNICOL PK 1978 Determination of specific radioactivity of plant aminoacids using dansylation. Anal Biochem 85: 71-78

12. MACNICOL PK 1983 Differential effect of sulphur deficiency on the composi-tion of the aminoacyl-tRNA and free amino acid pools of the developingpea seed. FEBS Lett 156: 55-57

13. MACNICOL PK 1983 Developmental changes in the free amino acid pool andtotal protein amino acids ofpea cotyledons (Pisum sativum L.). Plant Physiol72: 492-497

14. MACNICOL PK1986 Analysis of S-methylmethionine and S-adenosylmethio-nine in plant tissue by a dansylation, dual-isotope method. Anal Biochem158: 93-97

15. MACNICOL PK, L BERGMANN 1984 A role for homoglutathione in organicsulphur transport to the developing mung bean seed. Plant Sci Lett 36: 219-223

16. MERTZ ET, H MATSUMOTO 1956 Further studies on the amino acids andprotein of sulfur-deficient alfalfa. Arch Biochem Biophys 63: 50-63

358 Plant Physiol. Vol. 83, 1987




17. MILLERD A, D SPENCER 1974 Changes in RNA-synthesizing activity and

template activity in nuclei from cotyledons of developing pea seeds. Aust JPlant Physiol 1: 331-341

18. RANDALL PJ, JA THOMSON, HE SCHROEDER 1979 Cotyledonary proteins in

Pisum sativum. IV. Effects of sulfur, phosphorus, potassium and magnesiumdeficiencies. Aust J Plant Physiol 6: 11-24

19. RANDALL PJ, CW WRIGLEY 1986 Effects of sulfur supply on the yield,

composition and quality of grain from cereals, oilseeds and legumes. AdvCereal Sci Technol 8: 171-206

20. ROGNES SE, PJ LEA, BJ MIFLIN 1980 S-Adenosylmethionine-a novel regu-lator of aspartate kinase. Nature 287: 357-359

21. STEWARD FC, F CRANE, K MILLAR, RM ZACHARIUS, R RABSON, D MARGOLIS1959 Nutritional and environmental effects on the nitrogen metabolism ofplants. Symp Soc Exp Biol 13: 148-176

22. THOMPSON JF, CJ MORRIS, RK GERING 1960 The effect of mineral supply onthe amino acid composition of plants. Qual Plant Mater Veg 6: 261-275

23. WALLACE W, J SECOR, LE SCHRADER 1984 Rapid accumulation of -y-amino-butyric acid and alanine in soybean leaves in response to an abrupt transferto lower temperature, darkness, or mechanical manipulation. Plant Physiol75: 170-175

24. WHITE BA, FC BANCROFT 1982 Cytoplasmic dot hybridization. Simple analysisof relative mRNA levels in multiple small cell or tissue samples. J Biol Chem257: 8569-8572

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