Cuttings - NCBI

9 downloads 0 Views 771KB Size Report
Aug 7, 1984 - control or IBA pretreatment solution and introduced into glass vials containing .... The IRA-induced increase in PA levels concom- itant with root ...
Plant Physiol. (1985) 79, 80-83 0032-0889/85/79/0080/04/$01 .00/0

Polyamines and Root Formation in Mung Bean Hypocotyl

Cuttings' II. INCORPORATION OF PRECURSORS INTO POLYAMINES Received for publication August 7, 1984 and in revised form May 1, 1985

RA'ANAN FRIEDMAN2, ARIE ALTMAN*, AND URIEL BACHRACH Department of Horticulture, The Hebrew University of Jerusalem, Rehovot 76100, Israel (R.F., A.A.); and Department ofMolecular Biology, The Hebrew University and Hadassah Medical School, Jerusalem, Israel (U.B.) ABSTRAC1 The incorporation of [l4qsginine and I'4qornithine into various polyamines was studied in mung bean ( Vigna radiata IL.] Wilczek) hypocotyl cuttings with respect to the effect of indole-3-butyric acid on adventitious root formation. Both '4Qujginine and I'4Cornithine are rapidly incorporated into putrescine, spermidine, and spermine, with similar kinetics, during 5- to 24-hour incubation periods. The incorporation of arginine into putrescine is generally higher than that of ornithine. The biosynthesis of putrescine and spermidine from the precursors, in the hypocotyls, is closely related to the pattern of root formation: a first peak at 0 to 24 hours corresponding to the period of root primordia development, and a second peak of putrescine biosynthesis at 48 to 72 hours corresponding to root growth and elongation. Indole-3-butyric acid considerably enhances putrescine biosynthesis in both phases, resulting in an increase of the putrescine/ spermidine ratio. It is concluded that the promotive effect of indole-3-butyric acid on putrescine biosynthesis, from both arginine and ornithine, supports the hypothesis that auxin-induced root formation may require the promotion of polyamine biosynthesis.

the addition of propylamino residues derived from S-adenosyl methionine (1, 4). In recent years, it has been demonstrated that PA. play a significant role in plant responses to hormonal and environmental stimuli, both with respect to growth and active cell division, and to stress and senescence (2, 3, 6, 12). Thus, induction of cell division and growth was studied recently in several plant systems and its close relationship with PA metabolism and biosynthetic enzymes has been demonstrated (14, 15, 17). In a previous study (10) we suggested that PA are involved in IBA-induced root formation in mung bean cuttings. This conclusion was based on the effect of exogenous compounds, including PA precursors and some analogs and metabolic inhibitors, and on changes in the titer of endogenous PA. This was recently corroborated by similar data (13) showing that IBA leads to enhanced levels of PA in the hypocotyl where roots are formed (although we could not show that exogenous Spm enhanced root formation). The present investigation was undertaken in order to elucidate the role of PA in auxin-induced root formation by studying the incorporation of L-arginine and L-ornithine into various PA. In the following, we show that these two precursors are rapidly incorporated into Put, Spd, and Spm, with similar kinetics, and that the induction of adventitious roots by IBA is preceded and accompanied by an increase in the incorporation.

MATERIALS AND METHODS Plant Material and Rooting Experiments. Mung bean (Vigna radiata [L.] Wilczek) seedlings were grown and harvested, and rooting experiments were performed, as described previously (10). Protein Determination. Protein in the extracts was determined according to Lowry et al. (16) after precipitation with 4% w/v HCl04 and hydrolysis in 1 N NaOH, as described previously (10). Precursor Incorporation and Analysis. Freshly prepared hypocotyl cuttings were pretreated with 50 jAM IBA (in I mM KH2PO4-Na2HPO4 buffer, pH 5.8) for 24 h, after which incorporation of precursors was performed; or they were transferred to buffer solutions for an additional 24- to 48-h period, as mentioned, followed by incubation in the incorporation solution. 'Supported by a grant from the United States-Israel (Binational) All control solutions consisted of 1 mM phosphate buffer (pH Agricultural Research and Development Fund (BARD). 5.8). For incorporation studies, cuttings were removed from the 2 This work was submitted in partial fulfillment of the requirements control or IBA pretreatment solution and introduced into glass vials containing 0.4 ml 1 mm phosphate buffer (pH 5.8) and 0.2 for Ph.D. degree (R. F.), The Hebrew University. 3 Abbreviations: PA, polyamines (including di- and polyamines); Put, juCi of L-[U-'4C]arginine monohydrochloride (Amersham, 345 putrescine; Spd, spermidine; Spm, spermine; ADC, L-arginine decarbox- mCi/mmol) or L-[U-'4C]ornithine hydrochloride (Amersham, 285 mCi/mmol) which covered the basal cutsurface. The cuttings ylase; ODC, L-omithine decarboxylase; IBA, indole-3-butyric acid. 80

The biosynthesis and function of naturally occurring PA3 have been studied in detail in mammalian tissues and in procaryotes (4, 7, 19), whereas relatively little is known about PA metabolism in plants. Where such information is available in plants, it is based mainly on data of PA levels and the activity of their biosynthetic enzymes, ADC and ODC (5, 8, 18), rather than on incorporation of PA precursors. It is well established that arginine and ornithine can serve as precursors for Put biosynthesis. In mammalian cells ornithine seems to be the only precursor, via the activity of ODC (4, 19), while in procaryotes and plants arginine and ornithine may contribute equally to Put biosynthesis, via the activity of ADC and ODC, correspondingly (1, 3, 12). In either case, Spd and Spm are then formed from Put by

POLYAMINE BIOSYNTHESIS IN BEAN CUTTINGS were allowed to absorb the solution (2-5 h) and then transferred to vials with buffer only for total incorporation periods of 5 to

24 h, as specified in each experiment. All incubations were carried out in a controlled growth chamber (24 ± 1C, 16 h photoperiod of cool-white fluorescent light at about 3500 lux). Following incorporation, the hypocotyls were washed twice in cold buffer solution, a basal 1 mm slice was excised and discarded, and the cuttings were separated into hypocotyls, epicotyls, and leaves. Free PA were extracted with 4% (v/v) HC104,

purified, dansylated, and identified using silica gel TLC, as described previously (10). The PA spots which corresponded to the markers, as well as the entire silica of each lane, were removed, extracted with 10 ml toluene scintillation cocktail (1%, w/v, POPOP; and 0.05%, w/v, PPO) and counted in a Packard Tri-Carb liquid scintillation counter. Counting efficiency was about 90%, values were corrected for dpm and expressed on fresh weight or protein basis. In some experiments dansylated PA were chromatographed on Kodak 13179 chromatogram silica gel sheets with the same solvent system, and autoradiographed with x-ray film following exposure for 4 to 6 weeks at 4°C. Data are of a representative experiment, and consist of the mean of four points (two independently incubated samples of five cuttings each x two separate dansylations and TLC), with an average standard deviation of 12%. In some cases (Figs. 2 and 3) data were analyzed by Duncan's multiple range test. Experiments were repeated two to four times, yielding similar data. RESULTS The general pattern of incorporation of label from L-[U-'4C] arginine into various dansylated compounds of mung bean hypocotyl cuttings is demonstrated in Figure 1. About 55% of the total label extracted in HCl04 was incorporated into Put and Spd during 5 h incubation of freshly-excised cuttings, and only trace amounts were detected in Spm. Pretreatment of cuttings for 24 h in buffer resulted in reduced incorporation under these conditions. Label was detected also in dansylated products other 0

OPut

0

1iC ARGININE

30.1

e

016.0

c0 47.8

Ca

Q

4 Spd

d (3

c

(

24.6

0

16.8

0) 6.5

Sp

b a

Table I. Effect of IBA on the Incorporation of L-[U-'4CJArginine and L-[U-'4CJOrnithine into Polyamines in Freshly Prepared Mung Bean Hypocotyl Cuttings Cuttings were allowed to incorporate radioactive precursors for 5 h, in the presence or absence of 50 Mm IBA, as specified under "Materials and Methods". The ratio of Put to Spd was calculated from the dpm data. Per cent incorporation refers to the dpm detected in the Put + Spd + Spm calculated as per cent of the total dpm applied to the chromatogram as dansylated compounds. Data are of a representative experiment, with two replications of 5 cuttings each x 2 dansylations (see "Materials and Methods"). Put Spd Spm Put/Spd Incorporation dpm- mg-' ratio % protein

['4C]Arginine Control IBA

765 412

336 240

28 19

2.3 1.7

71 67

513 409

246 165

39 11

2.1 2.5

66 74

['4C]Ornithine Control IBA

Table II. Effect of a 24-h Period of IBA Treatment on the Incorporation of L-[U-'4CJArginine and L-[U-'4CJOrnithine into Polyamines in Mung Bean Hypocotyl Cuttings Cuttings were pretreated for 24 h with or without 50 AM IBA, followed by a 24-h period of incorporation of radioactive precursors, as specified under "Materials and Methods". The Put/Spd ratio and per cent incorporation were calculated as in Table I. For statistical treatment see Table I and "Materials and Methods". Put Spd Spm Put/Spd Incorporation dpm *mg'r ratio protein

['4C]Arginine Control IBA

1726 2388 2386 1440

239 0

0.7 1.7

42 32

450 2534 907 827

1404 257

0.2

14 27

['4C]Ornithine

9

f

81

O

STANARD

O

0

0-5 CONTROL

0-24 CONTROL

0-24 hr IBA

FIG. 1. TLC and autoradiography of dansylated products of mung bean hypocotyl cuttings. Cuttings were incubated in the incorporation solution with L-[U-'4C]arginine for 5 h either immediately after preparation of the cuttings (0-5), or following a 24-h pretreatment in phosphate buffer (0-24) with or without 50 gM IBA. The plate was exposed for 5 weeks, and the position of the various spots, including standards, is indicated (redrawn from the original chromatogram). The figures represent the per cent label in each spot calculated as per cent of the total dpm applied to the chromatogram as dansylated compounds.

Control IBA

1.1

than Put and Spd, both slow and fast-moving, which were not identified at this stage. To evaluate the interrelationships of IBA-induced root formation and polyamine biosynthesis, the incorporation of both LQU-'4CJarginine and L[U-'4C]ornithine was studied with respect to IBA pretreatment in freshly prepared cuttings (Table I) and following an extended IBA induction period (Table II). Label from arginine and ornithine was incorporated into PA within 5 h, the incorporation of arginine into Put and Spd being somewhat higher, and over 65% of the total label in the dansylated compounds was detected in the three major PA (Table I). IBA inhibited the incorporation of label into Put, Spd, and Spm, from both precursors, during the initial 5 h after preparation of cuttings. When incorporation of precursors was carried out for 24 h, after an initial 24-h period of IBA treatment, IBA significantly promoted the incorporation into Put and inhibited label accumulation in Spd, resulting in much higher Put/Spd incorporation ratios as compared with control cuttings (Table II). It is also obvious that only 14 to 42% of the total label was detected in the three PA when the incorporation was continued for 24 h. These data indicated that PA biosynthesis may depend on the -time after preparation of the cuttings and, therefore, on the stage of root development; thus, the pattern of pulse-incorporation of label (5 h) into PA was followed at various times after cuttings had been treated with IBA, both for L-[U-'4Cjarginine (Fig. 2)

Plant Physiol. Vol. 79, 1985

FRIEDMAN ET AL.

82

Z

Lfl

bc~~~~~~~~~b

0

24

48d7

HOURS

FIG. 2. Incorporation of L-[U-'4C]arginine into PA in response to IBA treatment of mung bean cuttings. Cuttings were incubated with L[U-'4C]arginine for 5 h either immediately after preparation of the cuttings (0), or at the end of a 24-h pretreatment in 50 MM IBA (24) or after additional, post-IBA, periods of 24 h (48) and 48 h (72) in phosphate buffer. Control cuttings were treated with phosphate buffer throughout. Hypocotyl sections were extracted at the end of the 5-h incorporation, and the distribution of label incorporated into Put, Spd, and Spin is expressed as per cent of the total dpm applied to the chromatogram as dansylated compounds. Data are of a representative experiment, with two replications of 5 cuttings x 2 dansylations. Values followed by the same letter are not statistically different at the 5% level (Duncan's multiple range test. See also "sMaterials and Methods").

FIG. 3. Incorporation of L-[U-'4C]ornithine into PA in response to IBA treatment of mung bean cuttings. All details as for Figure 2.

and for L-[U-'4Cjornithine (Fig. 3). The pattern of incorporation of the two precursors generally agrees, showing a low incorporation into Spin which does not change with time after preparation of the cutting and IBA treatment, a medium incorporation into Put combined with an increase in incorporation with time, and a higher, biphasic, incorporation into Spd. A consistent promotive effect of IBA on Put biosynthesis is evident, both with arginine and ornithine incorporation, whereas the effect of IBA on incorporation into Spd is variable, depending on the nature of the precursor and the time following IBA treatment. Calculations of the incorporation ratio into Put and Spd (Put/Spd ratio) indicate the clear promotive effect of IBA on Put biosynthesis, relative to that of Spd (Fig. 4). HOURS

DISCUSSION In our previous study (10) we showed that IRA, which induces root formation in mung bean hypocotyl cuttings, brings about a considerable increase in polyamine levels at the site of root formation, and this increase preceded the actual growth of adventitious roots. The IRA-induced increase in PA levels concomitant with root formation was later reported by Jarvis et al. (13). It is, thus, possible that PA have a regulatory role, combined with auxins, in the early events of adventitious root formation, i.e. active cell division and initiation of primordia (11). This is supported by the present data showing that root formation is also accompanied by an increase in Put biosynthesis, as judged

FIG. 4. Effect of IBA on the incorporation of precursors into Put, relative to the incorporation into Spd, in mung bean hypocotyl cuttings. The Put/Spd ratios were calculated from the incorporation data of L-[U'4C]arginine and L-[U-'4C]ornithine of Figures 2 and 3, based on dpm/ mg protein for each treatment and time point.

from incorporation of arginine and ornithine. Indeed, active incorporation of arginine into Put and Spd is evident in freshly prepared cuttings and it declines in senescing cuttings (Fig. 1), unless IBA is present during the initial 24 h after preparation of cuttings (Table II). It should be noted, however, that IBA, in fact, inhibits incorporation of arginine, and to a lesser extent of ornithine, during the initial 5 h (Table I). Thus, it can be

POLYAMINE BIOSYNTHESIS IN BEAN CUTTINGS concluded that the effect of IBA on increased PA biosynthesis is not immediate (and/or direct?), but is related to later stages which involve cell division and growth. This is corroborated by other studies which point to the involvement of PA in cell division and growth (5, 6, 8, 12, 14, 17). The increased ratio of Put/Spd in IBA-treated cuttings (Table II) is also indicative of active cell division (4, 10). A comparison of the incorporation of arginine and ornithine (Tables I, II) shows that both precursors are incorporated into Put, although the incorporation ofarginine is relatively higher. This could indicate that both the ADC and the ODC biosynthetic routes are operating in cuttings during adventitious root formation, as discussed previously (1, 3). Since adventitious root formation involves distinct and ordered cellular events, the timing of the IBA-induced changes in polyamine biosynthesis seem crucial in this respect. The data presented in Figures 2 and 3, and as summarized in Figure 4, indicate that two phases of Put biosynthesis can be distinguished: the first during the initial 0- to 24-h period, and the second during the 48- to 72-h period, both in IBA-treated cuttings and in control cuttings. Thus, Put biosynthesis occurs in the organ where roots are forming and during the two phases of adventitious root formation: in the induction and initiation phases (024 h) and in the growth and elongation phase (48-72 h) (9, 11, and references therein). It should be mentioned that few roots are formed in mung bean cuttings also in the absence of IBA (10), and Put biosynthesis in such cuttings is lower than in IBAtreated cuttings where more roots are formed (10, Figs. 2 and 3). We therefore conclude that the IBA-induced increase in PA biosynthesis may be related to IBA-induced increase in root formation. The decrease of label incorporation into Put during the intermediate, 24 to 48 h phase, may indicate its turnover and/or degradation, preceding the subsequent growth phase, but this was not followed in the present work. While there is no clearcut explanation for the fact that the trend in Put biosynthesis is much more consistent than that of Spd (Figs. 2 and 3), it is nevertheless confirmed by previous observations that Put concentrations (but not necessarily Spd) are correlated with cell division (4, 1 1). In accordance with similar recent studies (12, 14, 15, 17), the present work shows that active cell division and growth, and the effect of plant hormones on them, are accompanied by significant changes in PA biosynthesis and cellular levels. However, causal relationships are difficult to establish, since even the use of metabolic inhibitors, which should deplete PA, results, occasionally, in erroneous interpretations, and total depletion of PA in plant tissues was not attained. Indeed, studies indicate that in some cases (e.g. aleurone/GA system, 15) the early action of plant hormones is not on PA metabolism, but that the levels of

83

PA may be crucial for the regulation of the hormone action. It seems, therefore, that although PA fulfill at least several criteria to be regarded as growth substances, they seem instead to regulate certain effects of known plant hormones, as discussed elsewhere (2, 3, 12). Acknowledgment-The skillful assistance and cooperation of Mrs. N. Levin is gratefully acknowledged.

LITERATURE CITED 1. ALTMAN A, R FRIEDMAN, N LEVIN 1982 Arginine and ornithine decarboxylases, the polyamine biosynthetic enzymes of mung bean seedlings. Plant Physiol 69: 876-879 2. ALTMAN A, R FRIEDMAN, D AMIR, N LEVIN 1982 Polyamine effects and metabolism in plants under stress conditions. In PF Wareing, ed, Plant Growth Substances 1982. Academic Press, New York, pp 483-494 3. ALTMAN A, R FRIEDMAN, N LEVIN 1983 Alternative metabolic pathways for polyamine biosynthesis in plant development. In U Bachrach, A Kaye, R Chayen, eds, Advances in Polyamine Research, Vol 4. Raven Press, New York, pp 395-408 4. BACHRACH U 1979 Metabolism and function of spermine and related polyamines. Annu Rev Microbiol 24: 109-134 5. BAGNI N, D FRACASSINI 1974 The role of polyamines as growth factors in higher plants and their mechanism of action. In Plant Growth Substances. Hirokawa Publ. Co., Tokyo, pp 1205-1217 6. BAGNI N, D FRACASSINI, P TORRIGIANI 1982 Polyamines and cellular growth processes in higher plants. In PF Wareing, ed, Plant Growth Substances 1982. Academic Press, New York, pp 473-482 7. COHEN SS 1982 The polyamines as a growth industry. Fed Proc 41: 30613064 8. DUMORTIER FM, HE FLORES, NS SHEKHAWAT, AW GALSTON 1983 Gradients of polyamines and their biosynthetic enzymes in coleoptiles and roots of corn. Plant Physiol 72; 915-918 9. ERIKSN NE 1974 Root formation in pea cuttings. 2. The influence of indole3-acetic acid at different developmental stages. Physiol Plant 30: 158-162 10. FRIEDMAN R, A ALTMAN, U BACHRACH 1982 Polyamines and root formation in mung bean hypocotyl cuttings. 1. Effect of exogenous compounds and changes in endogenous polyamines. Plant Physiol 70: 844-848 11. FRIEDMAN R, A ALTMAN, E ZAMSKI 1979 Adventitious root formation in bean hypocotyl cuttings in relation to IAA translocation and hypocotyl anatomy. J Exp Bot 30: 769-777 12. GALSTON AW 1983 Polyamines as modulators of plant development. Bioscience 33: 382-388 13. JARVIS BC, PRM SHANNON, S YASMIN 1983 Involvement of polyamines with adventitious root development in stem cuttings of mung bean. Plant Cell Physiol 24: 677-683 14. KYRIAKIDIs DA 1983 Effect of plant growth hormones and polyamines on ornithine decarboxylase activity during the germination of barley seeds. Physiol Plant 57: 499-504 15. LIN PCC 1984 Polyamine metabolism and its relation to response of the aleurone layers of barley seeds to gibberellic acid. Plant Physiol 74: 975-983 16. LOWRY OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275 17. MIZRAHI Y, YM HEIMER 1982 Increased activity of ornithine decarboxylase in tomato ovaries induced by auxin. Physiol Plant 54: 367-368 18. SMITH TA 1978 Plant amines In EA Bell, BV Charlwood, eds, Encyclopedia of Plant Physiology, Vol 8. Springer-Verlag, Berlin, pp 433-460 19. TABOR CW, H TABOR 1976 1,4-Diaminobutane (putrescine, spermidine and spermine). Annu Rev Biochem 45: 285-306