Planta (Berl.) 91, 1--7 (1970)

Light and the Transport and Metabolism of Indoleacetic Acid in Normal and Albino Dwarf Pea Seedlings D. A. Mom~is Department of Botany, University of Southampton, U.K. l~eeeived IN*ovember17, 1969

Summary. The patterns of transport and metabolism of IAA-2-14C applied to the apices of intact normal and albino dwarf pea seedlings were essentially similar under given light conditions. Light greatly reduced the decarboxylation of the applied ]AA and stimulated the synthesis of indoleaspartic acid (IAAsp) in both normal and albino plants. In light considerably more 14C was exported from the apices of normal than albino plants; this result was attributed to the reduced capacity of the transport system in the latter. The specific activity of 14Cin the stem decreased logarithmically with increasing distance fl'om the treated apex. Light increased the steepness of the logarithmic profile. These results are discussed in relation to the rate of immobilization of IAA along the transport pathway by conversion to IAAsp. No evidence was found to support a previous suggestion (Pilet and Phipps, 1968) that IAA-oxidase activity and chlorophyll levels were causally linked. Introduction Several investigations have shown t h a t etiolated plant tissues m a y enzymically destroy indoleacetic acid (IAA) more readily than green tissues (Tang and Bonner, 1948; Galston and Baker, 1951; Gordon, 1954; Pilet, 1961; Hare, 1964). Recently Pilet and Phipps (1968) have demonstrated a reduction in the IAA-oxidase activity of roots of Lens culinaris Med., and leaves and whole seedlings of Nicotiana tabacum L. following their transfer from darkness to light. This loss of activity was associated with the appearance in extracts of these plant tissues of dialyzable compounds inhibitory to IAA-oxidase. Following the exposure to fight, chlorophyll synthesis occurred in the tissues, and it was suggested b y Pilet and Phipps (1968) t h a t the appearance of the inhibitors of IAA-oxidase was causally linked to the presence and amount of chlorophyll. I n support of this suggestion they cite evidence for the formation of inhibitory polyphenols in tissues undergoing photosynthesis. However, while their results demonstrate a simultaneous increase in the levels of inhibitor and chlorophyll in the tissues upon exposm'e to light, they do not exclude the possibility t h a t these increases occurred independently. 1

P l a n t a (Berl.), Bd. 91

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D.A. Morris:

W o r k on t h e influence of l i g h t on t h e t r a n s p o r t a n d m e t a b o l i s m of IAA-2-1~C a p p l i e d to t h e apices of i n t a c t d w a r f p e a seedlings (Pisum sativum L. cv ' M e t e o r ' ) c o n f i r m e d t h a t I A A was m o r e r a p i d l y d e g r a d e d in dark-gTown p l a n t s (Nesling, 1969). To t e s t t h e p o s s i b i l i t y t h a t t h e slower d e g r a d a t i o n of I A A in t h e l i g h t - g r o w n p l a n t s m i g h t be c a u s a l l y l i n k e d to t h e presence of c h l o r o p h y l l t h e w o r k was e x t e n d e d to a s t u d y of t h e t r a n s p o r t a n d m e t a b o l i s m of I A A a p p l i e d to t h e apices of i n t a c t n o r m a l a n d albino m u t a n t d w a r f p e a seedlings.

Materials and Methods Chlorophyll-deficient albino mutants were found to occur in commercially available samples of Meteor at an approximate frequency of 1 in 300 plants. Three thousand seeds were sown in trays of Levington compost in a heated greenhouse maintained at about 23 ~ C. The emerging seedlings were examined frequently and any albino mutants found were transplanted to 4 inch pots of compost. The mutants were dearly distinguishable from normal plants within a few hours of emergence by their characteristic yellowish pigmentation. Eight uniform seedlings were isolated in this way and were transferred with an equal number of normal seedlings to a growth cabinet maintained at 22.5 ~ C. Four plants of each type were exposed to light (photoperiod 16 hours; intensity 21,000 lux) and four were kept in darkness. Four days after transfer to the cabinet 0.25 txCi IAA-2-14C was applied to the apex of each plant as a 5.0 lzl droplet in 0.I % 'Tween 20' (specific activity of IAA 49 mCi per mM; ca 0.9 ~g IAA per plant). During application of the IAA darkgrown plants were briefly exposed to a dim green light source (Ilford gelatin filter No. 605, 'Spectrum Yellow-Green', 30 W incandescent source). The plants were returned to the cabinet for 7.5 hours and then dissected into individual organs (see Results below). The tissue was extracted overnight in darkness in 85% aqueous ethanol at 2~ C, and the residues extracted for two further periods of 4 hours in fresh 85% ethanol. Extracts and washings were combined, their radioactivity determined by liquid scintillation counting, and their composition examined by paper chromatography. Full details of the techniques of extraction, counting and chromatography have been given elsewhere (Morris, Briant and Thomson, 1969). Results and Discussion T h e t o t a l a c t i v i t y t r a n s p o r t e d from t h e apices was similar in n o r m a l a n d albino p l a n t s g r o w n in d a r k n e s s a n d in albino p l a n t s grown in fight; c o n s i d e r a b l y m o r e r a d i o a c t i v i t y was e x p o r t e d f r o m t h e apices of t h e l i g h t - g r o w n n o r m a l p l a n t s (Table 1). N o r m a l seedlings grown in l i g h t h a d a m u c h g r e a t e r fresh weight t h a n t h e e q u i v a l e n t albino p l a n t s (Table 2), a n d i t is possible t h a t t h e difference in t h e t o t a l a c t i v i t y e x p o r t e d from t h e apices of t h e two k i n d s of p l a n t in fight was m e r e l y a consequence of t h e difference in t h e c a p a c i t y of t h e t r a n s p o r t system. T h e v e r y similar specific a c t i v i t i e s of 14C i n t h e i n d i v i d u a l i n t e r n o d e s of t h e n o r m a l a n d albino p l a n t s in light s u p p o r t this view. There was little difference in t h e fresh weights of t h e n o r m a l a n d albino p l a n t s g r o w n in d a r k n e s s (Table 2), i n d i c a t i n g t h a t u n d e r these conditions g r o w t h p a t t e r n s in t h e two t y p e s of p l a n t were similar.

Light, Transport and Metabolism of IAA

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Table 1. Relative distribution and specific activities of ethanol-soluble radioactivity in normal and albino dwar] pea seedlings 7.5 hours after application of IAA-2-14C to the apices. Distribution is expressed as percentage of total activity exported from the apex; specific activities (in parentheses) in dpm per mg fresh weight. Activity applied was 670,290 dpm per plant Organ

Internode 4 Internode 3 Internode 2 Internodel Roots Cotyledons

Light-grown seedlings

Dark-grown seedlings

Normal

Albino

Normal

Albino

30.2 (349.7) 12.2 (68.0) 6.1 (38.1) 11.6 (25.5) 39.6 (8.3) 0.3 - -

27.4 (390.9) 12.0 (53.0) 7.1 (26.2) 10.9 (13.5) 41.6 (7.2) 1.0 - -

60.7 21.3 5.7 4.7 3.3 4.3

51.0 30.7 7.1 7.4 3.3 0.4

11,077

11,063

Total activity 28,709 exported from apex (dpm)

(59.7) (22.2) (12.6) (4.7) (0.4) --

(81.3) (36.5) (15.3) (9.2) (0.7) --

13,220

Table 2. Mean fresh weights of internodes a~ul root systems o/11-day old light- and darkgrown normal and albino dwarf pea seedlings (rag.plant -1) Organ

Internode 4 Internode 3 Internode 2 Internode 1 Roots

Light-grown seedlings

Dark-grown seedlings

Normal

Normal

Albino

112.5 106.1 50.1 111.6 897.6

83.2 111.3 61.3 106.7 642.5

24.8 51.6 46.3 130.1 1,376.0

Albino 7.6 25.1 30.3 89.9 640.6

The relative distribution of 14C between the individual organs of normal and albino plants in a n y one light t r e a t m e n t was strikingly similar (Table 1). I n the light-groin1 plants a considerable proportion of the total radioactivity exported from the apex was found in the lower internodes and roots, b u t in the plants kept for four days in darkness, little activity was transported b e y o n d the two u p p e r m o s t internodes during the 7.5-hour transport period. This effect was attributable to the greatly increased lengths of internodes 3 and 4 in the dark-grown plants. Specific activities of 14C, based on fresh weight measurements, were n o t influenced b y plant t y p e (Table 1). I n all t r e a t m e n t s the specific activity of laC in the stem decreased with increasing distance from the labelled apex. A l t h o u g h no length measurements were m a d e in the present experiment, previously unpublished d a t a provided b y Miss F . A . V . Nesling for normal Meteor seedlings grown in continuous light or complete darkness, 1"

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D.A. Morris:

1.0-

0

I

I

50 100 mm Distance from treatedapex

150

Fig. 1. Relationship between log10specific activity (dpm. mg dry wt-1) and distance from apex in seedlings of P. sativum treated with 0.92 Fg IAA-2-14C l~er plant (423,748 dpm per plant). The transport period was 7.5 hours. Regression equations. Dark-growa plants (solid symbols): Y = 3.4027--0.0924 X; Light-grown plants (open symbols) : Y = 3.7105--4).1002 X indicated that under both conditions of illumination there was a negative linear relationship between the logarithm of specific activity of laC in the stem 7.5 hours after labelling with IAA-2-14C and distance from the apex (Table 3 ; Fig. 1). The gradient was considerably steeper in the case of the fight-grown plants. These data suggest that the transport of radioactivity through the stem in the system under study closely resembled the model system described b y ]=[orwitz (1958} in which irreversible removal of activity from the transport stream leads to the establishment of a logarithmic gradient in the concentration of radioactivity along the transport pathway. That irreversible immobilization of auxin can indeed contribute to a logarithmic decline in radioactivity with increasing distance from donor agar blocks containing IAA-I-I~C, has been demonstrated in the more comprehensive studies of transport in A v e n a eoleoptiles by Goldsmith and Thimann (1962). Our previously published data indicated

Light, Transport and Metabolism of IAA

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Table 3. Influence of light on the growth o] Meteor dwarf pea seedlings and translocation of IAA-2-14C applied to the stem apices. Growth data arc based on samples of 10 plants (S. E. of mean shown in parenthesis); translocation data are means of 30 plants in each treatment (3 replications, each o] 10 plants), lnitial activity applied was 423,748 dpm per plant (0.92 #g I A A per plant) and the transport period was 7.5 hours. Treatments were applied 9 days after planting

Organ

Mean dry wt (mgTlant -1)

Apex Internode 4 Internode 3 Internode 2 Internode 1 t~oots Cotyledon

5.80 (0.664) 0.58 (0.110) 1.82 (0.126) 2.13 (0.089) 5.50 (0.571) 33.97 (2.490) 87.44 (6.609)

Apex Internode 4 Internode 3 Internode 2 Internode 1 Roots Cotyledon

5.88 (0.505) 7.05 (0.568) 13.93(0.583) 6.99 (0.921) 9.20 (0.922) 20.34 {1.130) 66.63 (6.347)

Mean length Mean (ram-plant -1) activity (dpm.plant-1)

Mean specific activity (dpm-mg-1)

Light-grown seedlings

-2.9 4.7 3.9 8.1 ---

(0.26) (0.33) (0.23) (2.05)

321,290 3,333 1,758 930 1,028 6,634 0

55,395 5,746 966 437 187 195 0

Dark-grown seedlings

-30.3 (2.52) 53.2 (1.94) 22.4 (0.96) 27.0 (1.05) ---

294,520 7,140 2,360 123 50 649 4

50,088 1,013 169 18 5 32 0

t h a t the principal loss of activity from the transport stream in pea epicotyls occurred as the result of conversion of I A A to indoleaspartic acid (IAAsp), which was shown to be immobile (Morris, Briant and Thomson, 1969). Clearly, the characteristics of the transport system will be influenced b y the rate of conversion of IAA to IAAsp, and factors leading to an increased rate of formation of IAAsp might be expected to increase the slope of the logarithmic gradient of total radioactivity in the stem. Observations reported below indicate t h a t a much more rapid conversion of I A A to IAAsp takes place in light t h a n in darkness, and it is suggested t h a t the observed differences in the slopes of the semi-logarithmic plots in Fig. 1 result primarily from ~his effect. However the situation is complicated b y the fact t h a t auxin destruction also plays a part in the loss of I A A from the transport stream. Using IAA-2-14C, we have found t h a t a major product of the decarboxylation of I A A (tentatively identified as iadole~idehyde) is also readily transported (Morris, Briant an4 Thomson, 1969). This

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D.A. Morris:

Table 4. Recovery o/14C-labelled I A A , I A A s p and decarboxylation products o] I A A (?IAld) in extracts of the stems of normal and albino seedlings of dwarf pea. The results are expressed as the percentage of the total activity recovered from each organ in 85% aqueous ethanol

Internode

Experimental conditions

Normal seedlings

4

Dark Light

63.7 83.6

3

Dark Light Dark Light Dark Light

2 1

IAAsp

IAA

Albino seedlings ?IAld

IAAsp

IAA

?IAld

5.3 4.4

13.2 4.4

47.0 82.4

19.5 1.7

17.4 7.1

32.1 68.7 23.6 58.9

19.0 4.3 14.2 8.6

22.9 8.5 29.1 14.3

30.6 75.7 20.0 59.3

21.5 4.5 9.9 10.3

13.2 6.7 42.1 14.5

17.3 54.7

9.7 1.6

43.1 17.7

41.6 50.7

5.1 6.9

37.7 16.7

product of auxin degradation will therefore be included in the t o t a l transported activity measured in the present experiments. The results of the chromatographic analyses of the ethanol-soluble extracts (Table 4) indicated t h a t the metabolism of IAA-2-14C in the stems of albino and normal plants was essentially the same. I n lightgrown plants of both types a major proportion of the activity present in the stems 7.5 hours after labelling was in the form of IAAsp. I n addition much smaller amounts of free I A A and deearboxy]ation products of IAA were also present in the stem. The proportion of the total activity attributable to the deearboxylation products increased towards the base of the stem. I n dark-grown plants IAAsp formed a much smaller proportion of the total activity in the stem and there were corresponding increases in the levels of both free I A A and the labelled degradation products of IAA. These observations are consistent with those of Lantican and Muir (1969) and of Nesling (1969), who found t h a t light promotes the formation of IAAsp in pea epicotyl and root tissues. I n dark-grown dwarf and normal cultivars of P . sativum Lantiean and Muir (1969) found no IAAsp and were unable to detect IAA-synthetase activity in preparations from tissues of such plants. Several observations indicate t h a t the formation of IAAsp can be induced by high levels of exogenous or endogenous IAA (Andreae and Good, 1955; Row, San/ord and Hitoheoek, 1961), and the concentration of IAA-2-1~C applied to the plants in the present experiments was probably sufficiently high to bring about the induction of the synthetase enzyme. The total amount of I A A applied (0.9~zg) was about 700 times greater than the amount

Light, Transport and Metabolism of IAA

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of endogenous diffusible auxin detected b y L a n t i c a n and Muir (1969) in apices of fight-grown dwarf and normal peas, and 3,000 times greater t h a n the a m o u n t detected in apices of dark-grown seedlings. I n the dwarf pea there appears to be no evidence for a chlorophylldependent inhibition of IAA-oxidase activity as suggested b y Pilet and P h i p p s (1968) for Lens roots and Nicotiana seedlings. Reduction of IAA-oxidase activity and stimulation of I A A s p synthesis b y light occurs equally in normal and chlorophyll-deficient albino plants. P a t t e r n s of translocation of the label from IAA-2-14C are identical in both types of plant under given conditions of illumination, and differences in the total a m o u n t of 14C t r a n s p o r t e d from the apices of normal and albino seedlings in light can adequately be explained in terms of the difference in the capacity of the transport system. The experiment demonstrates the occurrence of an inhibition of auxin degradation b y light whether or not the tissues contain chlorophyll and are capable of photosynthesis. The author is indebted to Professor S. H. Crowdy for helpful suggestions; to Miss F. A. V. Nesling for providing data used in Table 3; and to Mr. R. O'Prey for technical assistance. References Galston, A. W., Baker, R. S. : Studies on the physiology of light action. III. Light activation of a flavoprotein enzyme by reversal of the naturally occurring inhibition. Amer. J. Bot. 38, 199--195 (1951). Goldsmith, M.H.M., Thimann, K.V.: Some characteristics of movement of indoleacetic acid in coleoptfles of Arena. I. Uptake, destruction, immobilization, and distribution of IAA during basipetal translocation. Plant Physiol. 37, 492--505 (1962). Gordon, S. A. : Occurrence, formation and inactivation of auxins. Ann. Rev. Plant Physiol. 5, 341--378 (1954). Hare, R . C. : Indoleacetic acid oxidase. Bot. Rev. 80, 129--165 (1964). Itorwitz, L. : Some simplified mathematical treatments of translocation in plants. Plant Physiol. 33, 81--93 (1958). Lantican, B.P., Muir, R.M.: Auxin physiology of dwarfism in Pisum sativum. Physiol. Plant. (Kbh.) 22, 412--423 (1969). Morris, D.A., Briant, R.E., Thomson, P. G. : The transport and metabolism of l~C-labelled indoleacetic acid in intact pea seedlings. Planta (BEE.) 89, 178--197 (1969). Nesling, F. A. V. : The fate of indole-3-acetic acid-2-1~C in light- and dark-grown pe~ seedlings. B. Sc. Diss., University of Southampton (1969). Pilet, P.-E.: Les phytohormones de croissanee. Paris: Masson & Cie. 1961. - - P h i p p s , J. : Inhibition of auxin catabolism in relation to the chlorophyll content of the tissues. Planta (Berl.) 80, 82--88 (1968). Tang, u M., Bonner, J. : The enzymic inactivation of indoleacetic acid. II. The physiology of the enzyme. Amer. J. Bot. 35, 570--578 (1948). Dr. D.A. Morris Department of Botany University of Southampton Southampton, S09 5NH, England