Plant Physiol. (1990) 93, 439-4450032-0889/90/93/0439/07/$01 .00/0

Received for publication September 7, 1989and in revised form January 6, 1990

Polyamines and Flower Development in theMale Sterile Stamenless-2 Mutant of Tomato

(Lycopersicon esculentum Mill.)1

1. Level of Polyamines and Their Biosynthesis in Normal and Mutant Flowers

Rajeev Rastogi2 and Vipen K. Sawhney*Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO

ABSTRACT

The levels of free putrescine, spermidine, and spermine, andthe activities of ornithine decarboxylase and s-adenosylmethio-nine decarboxylase were determined in the floral organs of thenormal and a male sterile stamenless-2 (sl-2/sl-2) mutant oftomato (Lycopersicon esculentum Mill.). Under the intermediatetemperature regime, all mutant floral organs possessed signifi-cantly higher levels of polyamines and enzyme activities thantheir normal counterparts. In the low temperature-reverted mutantstamens, the polyamine levels and the activity of PA biosyntheticenzymes were not significantly different from the normal. It issuggested that the abnormal stamen development in the sl-2/sI-2 mutant is, in part, related to elevated levels of endogenousPAs.

The diamine putrescine and the PAs3 Spd and Spm areprobably of ubiquitous occurrence in plants and appear toplay an important role in plant growth and development (6,10, 29, 30). Changes in PA levels and activities of PA biosyn-thetic enzymes have been implicated in a variety of plantgrowth processes such as breaking of dormancy (2), seedgermination (31), fruit development (5, 28), somatic embry-ogenesis (7), pollen embryogenesis (13), adventitious rootformation (9), delay of senescence (1), and adaptation tostress (10).The PAs also appear to be involved in the flowering proc-

esses of plants, i.e. the induction of flowering (4, 12) and thedevelopment of floral organs (18). In mature flowers, pistilscontain a higher level of free PAs than the other organs (21)and stamens and gynoecium are known to be rich in PA-

Supported by an operating grant A0562 from the Natural Sciencesand Engineering Research Council of Canada to V. K. S.

2 Present address: Section of Plant Biology, Cornell University,Ithaca, NY 14853.

3 Abbreviations: PA(s), polyamine(s); sl-2/sl-2, stamenless-2; ODC,ornithine decarboxylase; SAMDC, S-adenosylmethionine decar-boxylase; LTR, low temperature regime; ITR, intermediatetemperature regime; HTR, high temperature regime; Put, putrescine;Spd, spermidine; Spm, spermine; MGBG, methylglyoxal-bis(guanylhydrazone).

conjugates (4). The expression of male sterility also has beenassociated with changes in endogenous PAs (19, 20). In severaltobacco mutants with defects in the PA biosynthetic pathway,Malmberg and associates related the altered levels ofPAs withabnormalities in floral organs (14-16, 18).The role of PAs in flower development can also be inves-

tigated by analyzing the level of PAs and the activity of PAbiosynthetic enzymes in floral mutants; (a) with specific de-fects in organ development and (b) in which the developmentof floral organs can be manipulated experimentally. In thesingle gene, sl-2/sl-2 mutant oftomato (Lycopersicon esculen-tum Mill.), stamen development can be altered by planthormones and temperature conditions. The mutant producesabnormal stamens that contain nonviable pollen and bearnaked ovules (26). Mutant plants grown under low tempera-tures or treated with gibberellic acid produce normal-lookingstamens that contain viable pollen, whereas those grown inhigh temperatures or treated with indoleacetic acid possesscarpel-like organs in place of stamens (25, 27). Thus, the sl-2/sl-2 mutant offers an excellent system for analyzing the roleof PAs in the differentiation of male and female organs.

In this paper, we report on the levels of free PAs and theactivities of ODC and SAMDC-biosynthetic enzymes ofPAs-in the mature floral organs of the normal and sl-2/sl-2mutant, and their relationship with the temperature-regulatedstamen development in the sl-2/sl-2 mutant. The role of PAsin the in vitro growth and development of normal and mutantfloral buds is the subject of the following communication.

MATERIALS AND METHODS

Seed Source and Plant Cultivation

The seed source and the maintenance of seed stock of thesl-2/sl-2 mutant and normal (+/+) line of Lycopersicon es-culentum Mill. was similar to that described earlier (25). Seedswere germinated in peat pellets ('Jiffy Sevens') and seedlingswith 3 to 4 leaves were transferred to 15 cm plastic potscontaining a mixture of peat, sand and loam (1:1:1 by weight).Plants were initially grown in the greenhouse with supple-mental lighting provided by cool-white fluorescent tubes andincandescent bulbs for 16 h/d. Subsequently, young plantswere transferred to controlled growth chambers maintained

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RASTOGI AND SAWHNEY

at the following day/night TR: low, 1 8°C/1 5°C (LTR); inter-mediate, 23/18°C (ITR); high 28/23°C (HTR). The lightingin the chambers was provided by Gro-lux wide-spectrumtubes at an intensity of 180 ,uE s-' m2, for 16 h/d. Plantswere fertilized twice a week with 20-20-20, a commercialfertilizer (Plant Products Co., Bramalea, Ontario). Matureflowers from different temperature conditions were used forpolyamine analyses and enzyme activity estimations.

Extraction and Determination of Polyamines

The various floral organs were homogenized in cold 5%perchloric acid at 100 mg/0.4 mL, and the extracts were kepton ice for 1 h. The homogenate was centrifuged for 10 minat 10,000g and the clear supernatant used for dansylationfollowing the modified method of Fores and Galston (8). To

2500 1 v I

2000

1500

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a)

0.C*

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1000

500

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45004000350030002500200015001000500

09onn&.WW

Spermine

1500 *

1000

500

.._f.F._%u1\"bl I.I..V e -09 Q(b 46, \0C3 0c3 9$.

-k,OFigure 1. Levels (nMol/g fresh weight) of Put (A), Spd (B), and Spm(C) in the floral organs and young leaves of the normal and sl-2/sl-2mutant. Values presented are the means. * Indicates that the valueis significantly different from the normal at P = 0.05. n = 12 in eachcase. Vertical bars represent SE.

the 0.2 mL supernatant, 0.4 mL dansyl (5-dimethylamino-naphthalene-l-sulfonyl) chloride (5 mg/mL in acetone andfreshly prepared), and 0.2 mL saturated sodium carbonatewere added. Each sample was mixed for 30 s and incubatedat 60°C for 1 h. Excess dansyl reagent was removed by treatingthe reaction mixture with 0.1 mL of proline (100 mg/mL)and incubating it for 30 min in the dark at room temperature.The dansylated polyamines were extracted twice with 0.5 mLbenzene. The organic phase was collected and stored at -20°Cuntil further use. Standards in the range of 200 to 1000 ,molof Put, Spd, and Spm also were dansylated either alone or incombination. Twenty gL of dansylpolyamines were loadedon 10 x 10 cm high performance TLC plates (Silica gel60, E. Merck, Darmstadt) and separated in chloro-form:triethylamine (5:1, v/v). The fluorescence of the dansyl-polyamines was measured by scanning the TLC plates with a

Quick Scan R and D densitometer (Helena Laboratories)using a 505 nm filter (excitation at 366 nm) and quanti-fied by comparing the areas under the peaks with those ofstandards.The identification ofunknowns was achieved by comparing

the RF values with those of the dansylated standards, and byspiking of the plant extracts with known amounts of a stand-ard. In the latter, the resultant increase in the peak area

confirmed the identification. Put was also identified by MS.

Enzyme Assays

For the measurement of ODC and SAMDC activity, 100mg of tissue was homogenized in 0.4 mL of 100 mm Tris-HCI buffer containing 0.1 mM EDTA, 1 mM DTT, and 0.1mM pyridoxal phosphate. The pH ofthe extraction buffer was8.0 for ODC and 7.6 for SAMDC. The homogenate was

centrifuged at 10,000g for 15 min at 4°C, and the supernatantused for enzyme assay.The activities of ODC and SAMDC were determined by

measuring the release of labeled '4CO2 from L-['4C]ornithineand S-adenosyl-L-['4C]methionine, respectively. For ODC ac-tivity, the reaction mixture consisted of 20 ,uL of enzymeextract, 80 ,L of extraction buffer, and 10 ,L of 10 ,uCi/mLL-["4C]ornithine (61 mCi/mmol) diluted in 0.2 mm unlabeledL-ornithine. For SAMDC activity, 10 ,uL of 10 ,uCi/mL of S-adenosyl-L-['4C]methionine (59.3 mCi/mmol) diluted in 0.2mm unlabeled S-adenosylmethionine was used. Each reactionwas carried out in triplicate in 12 x 75 mm disposablepolystyrene tubes sealed with plastic caps. A 40 x 4 mmWhatman No. 2 filter paper strip, soaked with 25 ,uL of 2 NNaOH and transfixed with a pin through the cap, was usedto trap the "4C02 evolved. The tubes were incubated at 37°Cfor 45 min and the reaction stopped by the addition of 0.2mL of cold 10% perchloric acid. The assay tubes were furtherincubated at 37°C for an additional 45 min to allow equili-bration of 14C02 between NaOH-soaked collecting strip, thegas phase and the acidified reaction mixture in the tube. Basalrates of decarboxylation (control) were estimated with eitherno enzyme or the addition of perchloric acid at time zero.After equilibration, the NaOH strips were removed from theassay tubes and put into 10 mL of scintillation cocktail(Aquasol-2, NEN Research products) in disposable scintilla-tion vials. Radioactivity in the strips was quantitated by using

Spermidine * B

**Y/

* 1

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POLYAMINES IN THE NORMAL AND MUTANT TOMATO FLOWERS

2000

L-

a,)-C,

cnL..0

4-Ca)

EE

1500

1000

500

0800

600

400

200 t tI

Figure 2. Activities (dpm/mg fresh weight/h) of ODC (A) and SAMDC(B) in the floral organs of the normal and sl-2/s1-2 mutant. Valuespresented are the means. * Indicates that the value is significantlydifferent from the normal at P = 0.05. n = 9 in each case. Verticalbars represent SE.

a Packard Tri-Carb 2000 CA Liquid Scintillation Analyzer.Enzyme activities are expressed as dpm/mg fresh weight/h.Each experiment consisted of three replicates and was per-

formed at least three times. An analysis of variance wasperformed on the data (32), and the differences in the meanswere tested by Duncan's multiple range test.

Chemicals

Hydrochlorides of Put, Spd, Spm, L-ornithine, and S-ad-enosyl L-methionine, and dansyl chloride were obtained fromSigma Chemical Co. L-[4C]ornithine hydrochloride, and S-adenosyl-L-['4C]methionine were purchased from Amersham.

RESULTS

PAs and Their Biosynthetic Enzymes in Mature FloralOrgans of the Normal and s1-2/s1-2 Mutant Grown in ITR

PAs

Put, Spd, and Spm were present in all floral organs of boththe normal and mutant flowers. However, there were differ-ences in the relative concentration of the three PAs amongthe various floral organs, as well as between the two genotypes(Fig. 1).

In both lines, the level of Put and Spd was lowest in thesepals and petals, intermediate in the stamens, and highest inthe gynoecium (Fig. 1, A and B). The level of Spm showed a

different trend; in the normal flowers, it was maximum in thesepals, intermediate in the stamens and gynoecium, and low-est in the petals (Fig. IC). In the mutant, however, the levelof Spm was similar in sepals, petals, and stamens, but washigher in the gynoecium (Fig. IC). Accordingly, the level oftotal PAs (the sum of Put, Spd, and Spm) was maximum inthe gynoecium, intermediate in the stamens, and lowest inthe sepals and petals.Of greater interest is the fact that the floral organs of the sl-

2/sl-2 mutant possessed significantly higher levels ofPAs thanthe normal, with the exception of Spd in the sepals (Fig. 1,A-C). The Put content in sepals, stamens, and gynoecium ofsl-2/sl-2 flowers was about twofold, and in the petals aboutfourfold, higher than those in the normal flowers (Fig. 1A).The levels of Spd and Spm also showed a similar trend inmost floral organs (Fig. 1, B and C).The PA content of young leaves was also compared in the

two genotypes. The mutant leaves possessed significantlyhigher levels of Spd and Spm, but not of Put, than the normal(Fig. 1, A-C).

ODC

The ODC activity was highest in the stamens, intermediatein the sepals and gynoecium, and lowest in the petals of boththe genotypes (Fig. 2A). A comparison of the ODC activityin the normal and mutant flowers showed that all mutant

20004-a

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COEcocm

0~(00

C

Putrescine

1500

1000

500

03000

2500

2000

1500

1000

5000

Figure 3. Level of Put (nMol/g fresh weight) in the floral organs ofthe normal (A) and sl-2/s1-2 mutant (B) grown under different temper-ature conditions. Values presented are the means. For each floralorgan, same letters on the bars indicate no significant difference at P= 0.05. n = 12. Vertical bars represent SE.

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RASTOGI AND SAWHNEY

Spermidine stamens in which it was significantly higher in LTR than in2000 ITR or HTR (Fig. 3A). In the mutant flowers also, the level

Normal b b A of Put was not affected in the sepals, petals, and gynoecium1500 L B ITR by different temperatures (Fig. 3B). However, in the stamens

U) aaLTR grown under different temperatures, there was a dramaticEaZ HTR change in the level of Put with the change in stamen mor-iooo 1000Pphology. In LTR, the mutant produces normal-looking sta-

SC >//mens (25) and in such stamens, the level of Put was signifi-coL a a cantly reduced and was comparable to that of normal stamens

r aa5 b0 in LTR (cf Fig. 3B, with 3A). In HTR, the mutant stamens

E | 1 |are carpel like and contain no pollen (25), and in this case the

cE 0 - - level of Put increased significantly and was not different from4500 Mutant that ofthe mutant gynoecium grown in different temperatures4000 asa-2s-2a (Fig. 3B).

a) 3500 The three temperature regimes did not affect the level of0.3000 _ c 8Spd in the sepals and stamens of normal flowers. However,000 C in the petals of HTR-grown flowers and in the gynoecium

_ 2500 from both LTR- and HTR-grown flowers, the Spd level was0 2000 greater than that in ITR (Fig. 4A). In the mutant, the Spd

1500 alevel was not affected by different temperatures in the various

C 1000a a a a a a b Z floral organs, except in stamens where it was significantly

500 reduced in LTR and dramatically increased in HTR (Fig. 4B).0 1 - The Spd level in HTR-grown mutant stamens was, however,

less than that in the gynoecium.ce o The level of Spm in the sepals and stamens of the normalline also was not affected by different temperatures (Fig. 5A).However, it was significantly higher in petals grown in LTR

Figure 4. Level of Spd (nMol/g fresh weight) in the floral organs of Sperminethe normal (A) and s1-2/s1-2 mutant (B) grown under different temper- 2000Normature conditions. Values presented are the means. For each floral Normal Aorgan, same letters on the bars indicate no significant difference at P +_ b=0.05. n = 12. Vertical bars represent SE. - 1500 ITR

U.) |121 LTRfloral organs possessed significantly higher level of activity (D HTRthan the normal (Fig. 2A). 1000 b

-C aa mc a aHSAMDC(1 50

SAMDC activity was also greater in the stamens than the LiiiL Jother floral organs of both the normal and sl-2/sl-2 mutant E 0(Fig. 2B). Further, the sepals, petals, and stamens of the 1sl-2s-2Mutant5Bnormal flowers possessed significantly lower SAMDC activitythan the mutant (Fig. 2B). In the gynoecium, however, bthe SAMDC activity was not different in the two genotypes Q 0 a 1(Fig. 2B). co a aa a a a

ciD aInfluence of Temperature on the Level of PAs and 0 bActivity of ODC and SAMDC in Floral Organs of the 2 500 bNormal and sl-2/sl-2 Mutant|

The level of PAs and the activities of ODC and SAMDCwere analysed in the normal and sl-2/sl-2 mutant flowers 0grown in low (LTR) and high (HTR) temperature regimes to e \

determine whether the temperature-regulated stamen devel- 5eopment in the mutant is associated with changes in the leveland biosynthesis of PAs.

Figure 5. Level of spermine (nMol/g fresh weight) in the floral organsPAs of the normal (A) and s1-2/s1-2 mutant (B) grown under different

temperature conditions. Values presented are the means. For eachIn the normal flowers grown under different temperatures, floral organ, same letters on the bars indicate no significant difference

the level of Put was not affected in most organs, except at P = 0.05. n = 12. Vertical bars represent SE.

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POLYAMINES IN THE NORMAL AND MUTANT TOMATO FLOWERS

and HTR, as well as in the gynoecium from HTR, than inITR (Fig. 5A). In the mutant, the level of Spm in sepals andpetals was not affected by the three temperature conditions(Fig. 5B). In the stamens, the level of Spm was decreased inLTR but unlike Put and Spd, it was not increased in HTR(Fig. 5B). In contrast, the Spm level increased in the HTR-grown mutant gynoecium.

ODC

ODC activity in most floral organs of the normal line wasnot significantly affected by the three temperature conditions,except in the gynoecium of LTR-grown flowers where it wasincreased (Fig. 6A). Similarly, in the mutant flowers, thedifferent temperature conditions did not significantly affectthe ODC activity in most organs, except the stamens in whichit was lower in both LTR and HTR than in ITR (Fig. 6B).

SAMDC

In the various floral organs of the normal line, SAMDCactivity was not affected by the three temperature regimes(data not presented). In the mutant, the SAMDC activity inthe sepals, petals, and gynoecium also was not affected bydifferent temperature conditions, but it was significantly re-duced in the stamens of LTR-grown flowers.

2000

-

*_r

a,)

c)

EEa

1500

1000

500

0

2000

1500

1000

500

0

Figure 6. ODC activity (dpm/mg fresh weight/h) in the floral organsof the normal (A) and s1-2/sl-2 mutant (B) grown under differenttemperature conditions. Values presented are the means. For eachfloral organ, same letters on the bars indicate no significant differenceat P = 0.05. n = 9. Vertical bars represent SE.

DISCUSSION

This study was conducted to investigate the possible role ofPAs in flower development of the normal, and male sterile sl-2/sl-2 mutant of tomato. The observations presented showthat there are differences in the levels ofPAs and the activitiesof their biosynthetic enzymes in the floral parts of the twogenotypes. Further, there is an apparent relationship betweenthe temperature-regulated stamen morphogenesis in the mu-tant and the PA levels and their biosynthesis. The results alsoshow that there are differences in the PA content and theactivities of PA biosynthetic enzymes among the variousorgans of a flower.The most significant finding of this study, however, is that

the floral organs ofthe sl-2/sl-2 mutant contain nearly doublethe amount of all PAs than the normal, with the exception ofSpd in sepals (Fig. 1, A-C). The difference was even greaterin the level of Put and Spm in the petals, and of Spd instamens and gynoecium. These observations suggest the pos-sibility that the sl-2/sl-2 mutation affects the biosynthesisand/or metabolism of PAs during flower development.The analysis of ODC and SAMDC revealed that in the

mutant, the activity of ODC in all floral organs and that ofSAMDC in sepals, petals, and stamens was significantly higherthan in the normal (Fig. 2, A and B). The greater activity ofthese two enzymes could account for the higher level of PAsin mutant flowers. However, the possible involvement ofother PA biosynthetic enzymes, e.g. arginine decarboxylase,as well as different metabolism of PAs in the two genotypes,which may contribute to different PA levels, cannot be ruledout.The regulation of stamen development in the sl-2/sl-2

mutant by temperature was also related to changes in PAlevels and the activity of PA biosynthetic enzymes. In themutant flowers grown in low temperatures, normal-lookingstamens and viable pollen are produced (25), and in suchstamens the level of the three PAs (Figs. 3B, 4B, and 5B) andthe activity of ODC (Fig. 6B) and SAMDC were lower thanin ITR-grown mutant stamens, but were similar to the PAlevels and enzyme activities in the normal stamens (clf withFigs. 3A, 4A, 5A, and 6A). In contrast, in high temperatureswhen the mutant stamens are carpel like (25), they show atwo-fold increase in the level of Put and Spd (Figs. 3B and4B) and the level of Put was comparable to that ofthe mutantgynoecium (Fig. 3B). The increase in Put and Spd titers inHTR-grown mutant stamens was, however, not associatedwith an increase in ODC or SAMDC activity and may berelated to some HTR-specific changes in PA biosynthesis ormetabolism. It should, nonetheless, be noted that the PAlevels and enzyme activities in most other floral organs ofboth the normal and mutant were not significantly affectedby different temperature conditions. Thus, the restoration ofmale fertility in the sl-2/sl-2 mutant by low temperatures isassociated with a decrease in PA levels and their biosynthesis,and the induction of carpelloidy by high temperatures withan increase in the levels of Put and Spd. Based upon theseobservations it can be argued that the elevated levels of PAsis likely one of the factors affecting stamen development inthe sl-2/sl-2 mutant. This suggestion is further supported by

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RASTOGI AND SAWHNEY

the fact that in normal flowers, which contain a lower PAlevel than the mutant, exogenous PAs induce abnormalitiesin stamens (24).The altered levels of PAs have been associated with abnor-

mal development of floral organs, particularly stamens andgynoecium, in other systems as well. Malmberg (14) reportedthat a tobacco mutant, which was male and female sterile,possessed lower Spd and Spm levels than the wild type.Another mutant of tobacco, Mgr3, selected for MGBG-resist-ance, possessed a higher Spd-to-Put ratio than the normal andwas also male and female sterile (15). In yet another MGBG-resistant mutant, Mgrl2 which is male sterile, purifiedSAMDC was highly resistant to MGBG suggesting that theMgrl 2 mutation may have altered the SAMDC protein (17).The genetic analysis of the Mgrl2 mutant revealed thatMGBG-resistance cosegregated with the male sterile pheno-type, implying that either the loci conferring the two traits areclosely linked, or that MGBG-resistance is caused by the samemutant allele as the shrunken male sterile, anthers. Recently,Gerats et al. (1 1) characterized the PA pathways in Petuniamutants with altered floral morphologies and found that onemutant line contained elevated level of Put as compared tothe wild type. In another study on Petunia floral mutants(Gerats, 1987; cited in Bernier, 1988), the level of Put, andthe activity of arginine decarboxylase and its mRNA, weretwo- to four-fold higher in mutant lines than the wild type.The analysis of PA-conjugates in the male sterile lines

provides another evidence for the possible involvement ofPAs in stamen development. In a study on the normal and acytoplasmic male sterile (F7T) line of corn, normal antherscontained high levels of PA-conjugates which were absent inthe sterile anthers (19, 20). In this male sterile line, therestoration of fertility by a restorer gene was correlated withhigh levels of PA-conjugates. Similarly, in some members ofAraceae, large amounts of hydroxycinnamic acid-PA conju-gates were present in the male fertile flowers, but not in themale sterile flowers (23). Thus, in these examples, the lack ofPA-conjugates was associated with male sterility. In the pres-ent study, the level of PA-conjugates was not determined.However, it is known that in solanaceous plants a largeproportion of total PAs occur as conjugates (28). Since thelevel of free PAs in the mutant flowers is higher than thenormal, it is possible that in the sl-2/sl-2 mutant, there isrelatively less conjugation of PAs which, as in the male sterilecorn, may be associated with abnormal pollen development.

This study has also shown that different organs of a flowercontain different levels of PAs and the activity of PA biosyn-thetic enzymes. In both genotypes, among the various floralparts, the gynoecium contained the highest total PA (Put +Spd + Spm), stamens the intermediate and sepals and petalsthe lowest content (Fig. 1, A-C). The activity of both ODCand SAMDC was, however, highest in the stamens in boththe normal, and sl-2/sl-2 mutant (Fig. 2, A and B). Highconcentrations of PAs and their conjugates in sex organs alsohave been reported in other cases. In Nicotiana, Put was themajor amine, and the stamens and gynoecium contained highlevels of Put and Spd (22). In Phaseolus vulgaris also, thepistils contained the highest PA levels and enzyme activitiesamong the various organs (21). Similarly, Martin-Tanguy et

al. ( 19) reported the presence of large amounts of specific PA-conjugates in the anthers and ovaries of Zea mays. Thepresence of relatively high levels of PAs in the stamens andgynoecium of tomato, and in other species, suggests that thePAs have a specific role in the development of sex organs.

In conclusion, this study strongly suggests the involvementof PAs in abnormal stamen development in the sl-2/sl-2mutant oftomato. However, additional studies on other floralmutants with defects in the PA pathway are warranted beforea physiological link between these substances and the regula-tion of flower morphogenesis and male sterility can be fullyestablished.

ACKNOWLEDGMENTS

R. R. acknowledges a University of Saskatchewan Graduate Schol-arship. The authors thank Dr. G. McKay, college of Pharmacy,University of Saskatchewan, for MS analysis of the polyaminesamples.

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POLYAMINES IN THE NORMAL AND MUTANT TOMATO FLOWERS

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