Plant Cell Reports

Plant Cell Reports (1982) 1 : 270-273

© Springer-Verlag 1982

Regulation of Abscisic Acid Metabolism in the Aleurone Layers of Barley Seeds Tuan-hua David Ho and Scott J. Uknes Department of Botany, University of Illinois, Urbana, IL 61801, USA Received June 25, 1982/October 25, 1982

ABSTRACT

MATERIALS AND METHODS

The regulation of metabolism of abscisic acid has been investigated in the i s o l a t e d aleurone layers of barley (Hordeum vulqare L.) seeds. The rate of conversion of abscisic acid to phaseic acid is enhanced by two to f i v e - f o l d when the tissue is pretreated with 10-5 M of this hormone. This enhancement can be observed with a pretreatment as short as two hours, and is prevented by t r a n s c r i p t i o n and t r a n s l a t i o n i n h i b i t o r s . The enhancement is accompanied by the appearance of new proteins which are induced by abscisic acid. I t is suggested that some of these ABA induced proteins are probably involved in the conversion from abscisic acid to phaseic acid. Abbreviations: ABA, abscisic acid; PA, phaseic acid; DPA, dihydrophaseic acid; SDS, sodium dodecyl sulphate; PAGE: polyacrylamide gel electrophoresis. INTRODUCTION Abscisic acid (ABA) has been known to regulate many physiological processes such as seed dormancy, l e a f senescence and the closure of stomates ( f o r review see Walton 1980). I t has been established that ABA can be converted in plant tissues into several metabolites including phaseic acid (PA) and dihydrophaseic acid (DPA) (Walton 1980). ABA can also form an ester or ether with a glucose molecule (conjugated ABA). Dashek et al (1979) as well as Ho (1979) have recently shown that exogenously applied PA was as e f f e c t i v e as ABA on the i n h i b i t i o n of m-amylase (EC 3 . 2 . ] . I ) synthesis, an enzyme related to germination in the aleurone layers of barley seeds. On the other hand, DPA did not possess significant biological activities. In order to investigate whether endogenous PA performs the same role as ABA, we have attempted to study the regulation of ABA metabolism in this tissue. In the current work we have found that pretreatment of barley aleurone layers with ABA activated the mechanisms converting ABA into PA.

0721-7714/82/0001/0270/$

01.00

Seeds of Himalaya barley (Hordeum vulgare L.) harvested in 1974 and 1979 were used in this work. ABA was purchased from Sigma Chemical Co. St. Lo~js,MO. Cis, trans-[G-°H]ABA(sp, ac. 33.2 Ci/mmole) and [ ~ S ] methionine (sp. ac. > 1,000 Ci/mmole) were obtained from Amersham, Arlington Heights, IL and New England Nuclear, Boston, MA, respectively. PA and DPA were g i f t s from Dr. Tom Scharkey, Plant Research Laborat or y , Michigan State University, East Lansing, MI. All the other chemicals were reagent grade. Aleurone layers were prepared and incubated as described by Ho et a l . (1981). For the extraction of ABA and i t s metabolites, aleurone layers were rinsed with 0.I mM cold ABA to exchange non-specifically bound radioactive ABA. The tissue was then homogenized in a mortar and pestle with 1.5 ml 90% methanol at 4 °C. The homogenate was centrifuged at 15,000 rpm in a Beckman J2-21 centrifuge at 20 °C f o r ten minutes. The supernatant was saved and the p e l l e t was re-extracted with 1 ml 90% methanol. Af t e r c e n t r i f u g a t i o n the extraction was repeated with 1 ml 90% ethanol at 60 °C. The supernatants were combined and dried in vacuum. The samples were analyzed by TLC on Whatman LKBDF preadsorbent plates. Usually 5 to 50 ~I of samples were applied to the preadsorbent area. The plates were developed with benzene: butanol: acetic acid (70:15:25) at room temperature. Af t er drying the plates were scraped at h a l f cm sections and the r a d i o a c t i v i t y in each section was measured in a s c i n t i l l a t i o n counter (Beckman LS 7500). The Rf values of the compound were determined by the travel distance on the main separation area of the TLC plate (the preadsorbent area was excluded). For the extraction of proteins the tissue was homogenized in the presence of SDS as described in Ho et al (1980). The proteins were analyzed by the SDS PAGE technique s i m i l a r to the procedure of Ho and Varner (1976) except slab gels were used in this work.

271 RESULTS When metabolites o f [3HI ABA i s o l a t e d from barley aleurone c e l l s were analyzed on TLC plates four d i f f e r e n t r a d i o a c t i v e regions were observed. The three less p o l a r regions, with Rf values of 0.64, 0.53 and 0.46, r e s p e c t i v e l y , we:me probably ABA, PA and DPA because they comigrated with a u t h e n t i c standards (Fig. l and 2). The most p o l a r region which was

18

A: Control 15

ABA

I2 o Conjugates "~

E

0

I

-6

-3

3

0

6

9

12

15

cm 18

B: Pretreoted wdh LO-SM ABA Conjugales 9

~

PA ABA

o x

S

o_ o

0 -6

I

I

-3

0

~-

3

6

I

12

15

cm

Fig. I. E f f e c t of pretreatment of barley aleurone layers with ABA on the metabolism of [3HI ABA. I s o l a t e d b a r l e y aleurone layers were incubated with or w i t h o u t ( c o n t r o l ) I0-5 M ABA f o r 24 h. A f t e r incubation fresh media c o n t a i n i n g l ~Ci [3HI ABA per ml were added and the tissue was f u r t h e r incubated for 8 h before ABA and i t s metabolites were analyzed by TLC as described under M a t e r i a l s and Methods. The p r o f i l e s of r a d i o a c t i v i t y on TLC plates are shown in this f i g u r e . S i m i l a r r e s u l t s have been obtained in at l e a s t three other experiments.

Fig. 2. Autoradiographic analysis o f metabolites of [3HI ABA separated on TLC p l a t e s . S i m i l a r to what was described under Fig. l except t h a t tissue was p r e - t r e a t e d with ABA for only 12 h. Autoradiography was performed by spraying the TLC p l a t e with Enhance (New England Nuclear), and exposing i t with XAR-5 x-ray f i l m (Kodak). ABA: pretreated with ABA. very close to the s t a r t i n g edge of the main s e p a r a t i o n area on TLC plates (see Materials and Methods) was most l i k e l y ABA conjugates and other polar m e t a b o l i t e s . The amount of [3HI PA extracted from tissues p r e t r e a t e d with lO -5 M ABA was two to f i v e f o l d higher than t h a t from tissues pretreated with b u f f e r only ( c o n t r o l ) (Fig. l and 2 ) . Since r a d i o a c t i v i t y , instead of chemical q u a n t i t y , was measured, the resul:t of this experiment i n d i c a t e s t h a t the rate of conversion from [3H] ABA to [3HI PA is enhanced in tissues p r e t r e a t e d with I0-5 M ABA. However, the same pretreatment with ABA did not a f f e c t the conversion from [3H] PA to [3HI DPA, the next m e t a b o l i t e in the pathway. The conversion o f [3HI ABA to the p u t a t i v e conjugates and other polar metabolites was also a f f e c t e d to a small e x t e n t depending on the length o f ABA pretreatment. The uptake of [3HI ABA was only r a r e l y influenced by ABA pretreatment. Thus, i t is apparent t h a t ABA can s e l f - i n d u c e i t s metabolic change to PA. A pretreatment of aleurone layers with I0-5 M ABA f o r as short as 2 h was s u f f i c i e n t to induce s i g n i f i c a n t enhancement on the conversion from [3H] ABA to [3N] PA (Table l ) . Table I .

Effects o f preincubation of ABA on the metabolism of [3H] ABA

Pretreatment time

Control ABA t r e a t e d cpm/fraction (% of t o t a l r a d i o activity)

2 h

ABA PA DPA Conjugates

2,955 578 533 2,138

(45%) (9%) (8%) (33%)

2,390.(32%) 1,490 (20%) 577 (8%) 2,706 (36%)

8 h

ABA PA DPA Conjugates

1,283 590 316 3,267

(22%) (I0%) (5%) (58%)

2,975 2,250 732 2,888

(32%) (25%) (8%) (31%)

272 Note :

Barley aleurone layers were pretreated with or without (control) 10-5 M ABA for 2 or 8 h. After pretreatment fresh media containing 1 ~Ci [3H] ABA per ml were added and the tissue was f u r t h e r incubated for 4 h before ABA and i t s metabolites were extracted and analyzed by TLC as described under Materials and Methodm Similar results have been obtained in at least three other experiments.

This enhancement was prevented in the presence of 0.I mM cordycepin (3'-deoxyadenosine) which i n h i b i t s the synthesis o f both poly (A) + and poly (A)- RNA in barley aleurone layers (Ho and Varner 1974), i n d i c a t i n g that the synthesis o f RNA is required in this process (Table 2). S i m i l a r l y , the protein synthesis i n h i b i t o r , cycloheximide, e f f e c t i v e l y blocked the conversion of ABA to PA suggesting the synthesis of new proteins is also necessary for this step. Both cordycepin and cycloheximide did not have any s i g n i f i c a n t e f f e c t on the uptake of [3HI ABA by aleurone layers. Table 2.

Effect of t r a n s c r i p t i o n and t r a n s l a t i o n i n h i b i t o r s on the metabolism of ABA in barley aleurone layers. Pretreatment -ABA (control)

+lO-5 M ABA

% of total radioactivity in tissue as pha~eic acid (PA) Control 8.0 20.3 (no inhibitor) + Cord%cepin (lO-~ M)

2.3

2.1

+Cycloheximide (I0 ~g/ml)

1.2

0.5

Note:

Barley aleurone layers were incubated under the variods conditions indicated above for 4 h. The tissue was then labeled in fresh medium containing i n h i b i t o r and [3HI ABA (l ~Ci/ml) for 2 h~ Abscisic acid and its metabolites were extracted and analyzed as described under Materials and Methods.

I t has been previously observed that ABA induces a group of new proteins in barley aleurone layers (Ho, 1979 and Jacobsen et al. 1979). We have now extended the above observation by i n v e s t i g a t i n g the kinetics o f synthesis of these ABA-induced proteins (Fig.3).The induction of these proteins was already quite apparent a f t e r 2 h incubation with 10-5 M ABA. This induction was also sensitive to RNA synthesis i n h i b i t o r s such as cordycepin. DISCUSSION The metabolism of ABA in barley aleurone layers is enhanced by ABA i t s e l f . However, this enhancement is l i m i t e d to the conversion from ABA to PA and, to much lesser extent, the formation of ABA conjugates. This enhancement is dependent on RNA and protein synthesis, and accompanied by the formation of new proteins which are apparently induced by ABA. Because of these coincidences we would l i k e to suggest that some of these ABA-induced proteins are probably involved in

Fig. 3. Effect of ABA on the induction of new proteins. Isolated barley aleurone layers were incUbated with lO-5 M ABA for different lengths of time ( h ) as indicated in the figure. The tissue was then labeled with [35S] methionine (25 ~Ci/ml) for 2 h before proteins were extracted and analyzed as described under Materialsand Methods. Equal amount of total radioactivity was intended for each channel on the gel. The autoradiogram of the SDS gel is shown in this figure. The arrows point to two of the most apparent ABA~induced proteins with m.w. of l l 5 Kd (upper) and 27 Kd (lower), respectively. Similar results were obtained in another experiment. the s e l f - i n d u c t i o n of ABA metabolism. In other words, our observation is analogous to the substrate induction of enzymes such as n i t r a t e reductase induction by n i t r a t e ( F i l n e r et al. 1969). However, ABA, at the concentration o f 10-5 M, is s u f f i c i e n t to induce i t s own metabolismby s e v e r a l - f o l d , while n i t r a t e reductase induction requires a concentration of n i t r a t e at 10-3 M or higher. At least two lines of potential physiological significance of this self-induction of ABA metabolism exist. First, this canserve as a scavenger mechanism which removes excessive ABA. Second, ABA may have to be converted to PA to be biologically active. Again, this is similar to nitrate metabolism in plant tissues where nitrate has to be reduced to ammonium in order to be u t i l i z e d in the synthesis of nitrogencontaining compound. The observation that exogenous PA but not DPA is effective on the inhibition of ~-amylase synthesis (Dashek et al. 1979 and Ho 1979) is in accordance with the second p o s s i b i l i t y .

273 REFERENCES Dashek WV, Singh BN, Walton DC (1979) Plant Physiol. 64:43-48 F i l n e r P, Wray JL, Varner JE (1969) Science 58: 1520-1525 Ho THD (1979) Plant Physiol. 63:s444 Ho THD, Varner JE (1976) Plant Physiol. 57:175-178 Ho THD, Varner JE (1974) Proc. Nat. Acad. Sci. 71: 4783-4786 Ho THD, Nolan RC, Shute DE (198i) Plant Physiol. 67:I026-I031

Ho THD, Shih SC, Kleinhofs A (1980) Plant Physiol. 66:153-157 Jacobsen JV, Higgins TJV, Zwar JA (1979) In: Rubenstein I , P h i l l i p s RL, Green CE, Gengenbach BG (ed) The plant seed: development, preservation and germination, Academic Press, New York, pp 241-262 Walton DC (1980) Ann. Rev. Plant Physiol. 31: 453-489