Dec 21, 1983 - SCOrr J. UKNES AND TUAN-HUA DAVID HO*2. Department ofPlant Biology, University ofIllinois, Urbana, Illinois 61801. ABSTRACr. As a part ...
Plant Physiol. (I1984) 75, 1126-1132 0032-0889/84/75/11 26/07/$0 1.00/0
Mode of Action of Abscisic Acid in Barley Aleurone Layers' ABSCISIC ACID INDUCES ITS OWN CONVERSION TO PHASEIC ACID Received for publication December 21, 1983 and in revised form March 20, 1984
SCOrr J. UKNES AND TUAN-HUA DAVID HO*2 Department ofPlant Biology, University ofIllinois, Urbana, Illinois 61801 ABSTRACr As a part of our effort to study the mode of action of abscisic acid (ABA) and its metabolites during seed germination, we have investigated the regulaton of ABA metabolism in barley (Hordeum vulgare) aurone layers and a few other plant tissues. The rate of conversion of pHIABA to I3Hlphaseic acid (PA) the first stable metabolite of ABA, is enhanced by 2- to 5-fold in barley aleurone layers when the tissue is pretreated with ABA. However, the conversion of I3HIPA to 1HI dihydrophaseic acid (DPA), the next metabolite after PA, is not enhanced by pretreatment with either ABA or PA. The ABA enhancement of its own metabolism in barley aleurone layers is detectable with a pretreatment of ABA rai from 109 to 10 molar. This apparent self-induction of ABA conversion to PA can be observed after the barley aleurone layers have been treated with 10 molar ABA for as short as 2 hours, and is inhibited by the transcription inhibitor, cordycepin (3'-deoxyadenosine), or the translation inhibitor, cycloheximide. The self-induction of ABA conversion to PA also occurs in wheat aleurone layers, but not in other plant tissues that have been investipted, including corm root tips, barley embryos, barley, and soybean leaf discs. It is probably a phenomenon unique to the aleurone layers of some cereal grains. In view of the recent observations that ABA is able to induce new proteins in barley aleurone layers, we suggest that some of these ABA-induced proteins are involved in the conversion from ABA to PA in this tissue.
It has been well documented that ABA exerts regulatory roles in many physiological processes such as stomatal closure, bud dormancy, seed dormancy, seed development, and germination (for a review, see 17). The mode of ABA action during seed germination has been studied in many systems including cotton embryos (4, 12), wheat embryos (1), and barley aleurone layers (2, 8, 10, 13, 18). In barley aleurone layers, ABA inhibits the GA3-enhanced synthesis of a-amylase (2, 10). This inhibition is not due to the direct competition between GA and ABA for a common site of action because high concentrations of GA do not completely overcome the effect of ABA (2, 13). Similar to what was reported in cotton and wheat embryos (1, 12), the effect of ABA in barley aleurone layers can be prevented by transcription inhibitors (e.g. cordycepin) indicating that the action of this hormone is dependent on the continuous synthesis of RNA and/or proteins (10). More recently, it was discovered
that ABA induces the synthesis of several new proteins in both cotton embryos (4) and barley aleurone layers (8, 9, 11, 15). The function of these ABA-induced proteins is still unknown. Dashek et aL (3) have partially determined a pathway of ABA metabolism in barley aleurone layers. As in other plant tissues ABA is first converted to PA, then to DPA, which is further metabolized to unidentified polar compounds. In addition, ABA, PA, and DPA can form conjugates with glucose via an ester linkage. Dashek et al. (3) and Ho (8) have examined the biological activities of isolated PA and DPA. It was observed that PA is at least as active as ABA in inhibiting the GA-enhanced synthesis of a-amylase. However, DPA has little or no activity. Since exogenous PA is active in the inhibition of a-amylase synthesis in barley aleurone layers, it has become necessary to investigate the physiological role of endogenous PA. In this work we have studied the regulation of PA formation, and our observations indicate that ABA can induce its own conversion to PA. In contrast, the conversion from PA to DPA is not affected by either ABA or PA.
MATERIALS AND METHODS Chemicals. Cis, trans, and mixed isomers of ABA were purchased from Sigma Chemical Co. DL-cis,trans-[3HJABA (10-20 Ci/mmol) and DL-cis,trans-`'4C]ABA (11.9 mCi/mmol) were obtained from Amersham. The purity of the labeled ABA was greater than 98% as analyzed by TLC. Phaseic acid and DPA were purified according to Sharkey and Raschke (16) and given to us by Dr. Thomas Sharkey, Michigan State University. All other chemicals were reagent grade. Preparation of Aleurone Layers.The embryos of barley seed (Hordeum vulgare cv Himalaya, 1979 crop) were excised with a dissecting knife. The endosperm half of the seeds (henceforth called half-seeds) were surface sterilized with 1% NaOCl (5-fold dilution of 'Clorox') for 15 min. The half-seeds were washed seven times with sterile-distilled H20. The half seeds were then incubated on a sterile sand plate (50 g of acid-washed sand in a 10-cm Petri dish) with 13 ml of 20 mm sodium succinate buffer (pH 5.0) containing 20 mM CaCl2 (henceforth called succinate buffer) for 3 to 4 d at room temperature in the dark. Wheat (Triticum aestivum L.) half-seeds were incubated under identical conditions for only I d. After incubation on the sand plate, aleurone layers were peeled from the imbibed half-seeds with the help of two spatulas. Ten aleurone layers were placed in a 25-ml flask containing 2 ml of succinate buffer and then shaken at 120 strokes/min. The aleurone layers were pretreated with ABA and labeled with [3H]ABA as illustrated in Figure 1. It is important to note that the chemical concentration of [3HJABA (3-30 nM) is much smaller than that of the nonradioactive ABA used in the pretreatment. Other specific incubation and labeling conditions are described later. Preparation of Barley Embryos. Barley seeds were surface sterilized as described previously. The seeds were then incubated
'Supported by National Science Foundation Grants PCM 80-21632 and 82-40868. 2 Cufrent address: Department of Biology, Washington University, St. Louis, MO 63130. 3Abbreviations: GA, gibberellin; PA, phaseic acid; DPA, dihydrophaseic acid. 1126
SELF-INDUCTION OF ABA CONVERSION TO PA
1127
1) ABA Pretreated Sample Pretreatment
Label i ng H)ABA 0.1 - 1 pCi /ml
IABA)
= 10 8 -o4 2 to 24 h
M
Wash
3-30 2nM h
>
Extraction and TLC
FIG. 1. Experimental design. 2)
Control Pretreatment
Buffer only 2 to 24 h
Wash
Labeling [3H] ABA 0.1 - 1 pCi/ml 3 230 nM
on two layers of filter paper moistened with 4 ml of 20 mm CaC12 containing various concentrations of ABA (0_10-4 M) for 0.5 or 1.5 d at 28C in the dark. The embryos were then removed from the endosperm, washed with succinate buffer, and placed in a 25-ml flask (four/flask) with 1 ml of buffer containing [3H]ABA (1 uCi/ml). The flasks were shaken (120 strokes/min) at 25C for 3 h before ABA and its metabolites were extracted and analyzed. Preparation of Corn Root Tips. Corn seeds (Zea mays L. var A632 x C1042) were surface sterilized for 1 min with 1% Na hypochlorite, and then rinsed seven times with distilled H20. The seeds were germinated according to the procedure of Gronewald and Hanson (7) and the roots were harvested on the 4th d. One-cm tips from primary roots were placed in a 25-ml flask with 2 ml of succinate buffer and preincubated in a shaker (120 strokes/min) for 4 h at 25C under room light. This pretreatment is to allow the tissue to recover from the excision injury (7). The root tips were further incubated for 5 h in succinate buffer with or without 10-' M ABA. The metabolism of ABA was studied by labeling the root tips with 1 gCi of [3H]ABA in 1 ml of fresh succinate buffer for 3 h. The extraction and analysis of ABA and its metabolites are described below. Preparation of Barley and Soybean Leaf Discs. Leaf discs of barley and soybean (Glycine max L.) were prepared by punching leaves of approximately 14-d-old plants with a No. 2 cork borer. Ten discs were placed in a 25-ml flask with 2 ml of succinate buffer, and shaken (120 strokes/min) at 25C for 4 h to allow the tissue to recover from the excision injury. The tissue was further incubated for 6 h with or without 10-5 M ABA before being washed and labeled with [3H]ABA (1 ACi/ml) for 4 h. The ABA and its metabolites were extracted and analyzed as described below. Isolation and Analysis of ABA and Its Metabolites. Tissues labeled with [3H]ABA were washed three times with 3 ml of 10' M ABA to remove nonspecifically bound [3H]ABA, and ground in a mortar with 1.5 ml of 90% methanol. The homogenate was centrifuged at 15,000g for 10 min in a Beckman J2-21 centrifuge equipped with a JA 17 rotor. The pellet was reground in I ml of 90% methanol, and then centrifuged as above. This second pellet was further extracted with 90% ethanol at 60C for 1 min by vortexing and centrifuging. All three supernatants were combined and taken to dryness in vacuum at room temperature. This procedure allows the extraction of more than 98% of the total radioactivity from the tissue. Although methanol was used
>
Extraction and TLC
in this procedure, we did not detect any artifactual formation of methyl ABA. The sample was then dissolved in 200 Ml of methanol. Five to 50 Ml of the samples were spotted on the preadsorbent area of a Whatman LK 6DF TLC plate. The plate was developed with benzene:butanol:acetic acid (70:15:25) at room temperature. After drying, the plate was scraped in 0.5-cm sections, and radioactivity in each section was determined in a Beckman LS 7500 scintillation counter using a toluene-based scintillation fluid. The peaks of radioactivity were identified by comparing their RF values with those of authentic ABA, PA, and DPA.
RESULTS Abscisic Acid Pretreatment of Barley Aleurone Layers Enhances the Formation of IHIPA. [3H]ABA and its metabolites extracted from barley aleurone layers could be separated into four peaks of radioactivity on TLC plates (Fig. 2). Peaks with RF values of 0.64, 0.53, and 0.46 comigrated with authentic ABA, PA, and DPA, respectively, in at least two different solvent systems. The DPA peak may contain both DPA and its isomer, epi-DPA, which can not be resolved in our TLC system. The radioactive region near the origin appeared to contain polar compounds derived from [3H]ABA. About 75% of the radioactivity recovered from this region can be alkaline hydrolyzed to yield ABA. Therefore, this region probably contains sugar conjugates of ABA as well as other unidentified polar metabolites of this hormone. Pretreatment of aleurone layers with 10-' M ABA for 12 h did not affect the uptake of [3H]ABA, yet it caused a 2to 5-fold increase in the relative amount of [3H]PA as compared to the control tissue preincubated in buffer only (Fig. 2). There was also a concomitant decrease in the relative amount of [3H] ABA in the ABA-pretreated tissue as compared to the control while the relative amounts of [3H]DPA in both samples was approximately the same. Therefore, it appears that the pretreatment of barley aleurone layers with ABA enhances the formation of PA but not of DPA. The radioactivity in the peak containing conjugates and other polar metabolites varied in different treatments. Thus, the exact effect of pretreatment of ABA on the amount of radioactivity in this peak is unclear to date. To determine whether the relative amount of radioactivity in ABA and PA peaks represent the rate of conversion from ABA to PA or just the accumulation of these compounds, we have conducted the following set of experiments. Barley aleurone layers were preincubated for 12 h with or without 10-5 M ABA.
1128
UKNES AND HO
Plant Physiol. Vol. 75, 1984 60
fA ABA
-
-
B. PA
I0
0-
-
,
., 20 I
> 60 20
-Iz
II4--4 IF--I--I -II
C. DPA
D.
40 0 -
o
Conjugates-
polar and metabolites
4
18
12
8 12 4 8 12 Hour of Labeling FIG. 3. Time course of labeling barley aleurone layers with [3H]ABA. Barley aleurone layers were preincubated in succinate buffer with or without 10-s M ABA for 12 h. The tissue was then labeled in 1 ml of succinate buffer with I uCi [3H]ABA for various lengths of time. ABA and its metabolites were extracted and analyzed as described under "Materials and Methods." The percentage of total radioactivity in individual peaks on TLC plates was plotted versus the length of labeling time. Control (@-4); ABA preincubation sample (O --- ).
B. +ABA
-
conjugates and polar metabolites
PA ABA
9 6
DPA
-3
0
3
6
9
12
cm
FIG. 2. Effect of pretreatment of barley aleurone layers with ABA on the metabolism of [3H]ABA. Barley aleurone layers were incubated with or without 10-s M ABA for 24 h. After incubation, fresh medium containing I gCi [3H]ABA/ml was added and the tissue was incubated for an additional 4 h before ABA and its metabolites were extracted and analyzed as described under "Materials and Methods." The cpm were graphed against the distance travelled in cm. The negative values on the distance travelled are the preadsorbent area on the TLC plate. 'O' is the junction between the preadsorbent area and the silica gel. The shaded area indicates the amount of [3H]PA. A, Control, no ABA pretreatment; B, +ABA, pretreated with 10-5 M ABA.
These layers were then washed and labeled with [3H]ABA for various lengths of time before ABA and its metabolites were extracted and analyzed. As shown in Figure 3B, the relative amount of [3H]PA was always higher in ABA-pretreated tissue than in the control. The increase of [13H]PA reached a maximum after 2 to 4 h of labeling. Therefore, in order to have a measurement close to the rate of PA formation, rather than the accumulation of this compound, we labeled the aleurone layers with [3H]ABA only for 2 to 4 h in all the other experiments. Another benefit of using short labeling times is that virtually no labeled ABA metabolites are secreted into the incubation medium. In addition, the results in Figure 3 show that the relative amount of [3H]ABA decreased continuously with time, while the [3H]PA level rose initially and then declined later, and [3H]DPA followed a continuous increasing trend. This kinetic information has confirmed the established ABA metabolic pathway, i.e. ABA -+ PA -. DPA. Again, the complexity of the peak continuing conjugates and other polar metabolites does not allow us to draw any useful information. Another problem we have encountered in this research is the possible expansion of the internal ABA pool size during the preincubation period. If the ABA pool size expands substantially, then the rate of conversion from ABA to PA would be underestimated in the ABA-pretreated sample. Therefore, in the following experiment we incubated 10 barley aleurone layers with 0.1 1gCi/ml [14C]ABA (8.4 x 1o-6 M) for various lengths of time, and estimated how much ABA was taken up by the tissue. As shown
in Table I, about 15% of the exogenous ABA was taken up by aleurone layers within 2 h of incubation, and up to 44% by 24 h. About 70% of the ABA taken up by the tissue during the initial 2 h remained as ABA, while after 24 h only 10% of the ABA remained unmetabolized. Based on the estimation that 10 aleurone layers contain 0.1 ml H20 (Ho, unpublished observation), our calculation indicated that the internal concentration of ABA in ABA-pretreated tissue is 9 x 101 M at 2 h, and 3.5 x 10-6 M at 24 h. It has been determined that the ABA content in the endosperm of mature barley grain is about I gg/I00 g of dry matter (6). Assuming endosperm water content is about 10% of fresh weight and there is an equal distribution of ABA among various parts of endosperm, we calculate that the concentration of ABA in aleurone layers to be about 4 x 10'- M. Comparing this value with the ABA concentration in the ABA-pretreated samples, it is apparent that there is a substantial increase in the ABA pool size due to the pretreatment. Therefore, the 2- to 5fold enhancement of PA formation we have observed appears to be an underestimation (see "Discussion"). Time Course and Dosage-Response Curve of the Effects of ABA Pretreatment of r3HPA Formtion. To determine how fast the formation of PA could be enhanced, barley aleurone layers were preincubated with or without ABA for various lengths of time before the metabolism of [3H]ABA were analyzed. We found that an ABA-preincubation for as short as 2 h could elicit an increase in [3H]PA formation. This effect continued to at least 20 h of preincubation (Figs. 4B and 5). In contrast, the relative amount of [3H]ABA decreased for most of the time course as the consequence of the same ABA pretreatment (Fig. 4A). Different from both ABA and PA, the formation of [3H]DPA remained virtually unchanged (Fig. 4C). As shown in Figure 4D, the formation of conjugates and other polar metabolites was decreased in the ABA-pretreated sample. However, in other experiments the formation of these compounds was enhanced by ABA-pretreatment. The cause of this variability was not pursued in this study. A dosage-response curve of the effects of ABA pretreatment was determined by preincubating barley aleurone layers with various concentrations (10-8 to I0 M) ofABA for 12 h followed by a 2-h labeling period with [3H]ABA. As shown in Figure 6B, the formation of [3H]PA was only slightly enhanced when the concentration of ABA in pretreatment increased from 108 to 10- M. However, concentrations of ABA higher than 106 M
1129
SELF-INDUCTION OF ABA CONVERSION TO PA
Table I. An Estimation of Endogenous ABA Concentration in ABA-Treated Barley Aleurone Layers Ten barley aleurone layers were incubated for various lengths of time in succinate buffer with ["C]ABA (0.1 sCi/ml, 8.4 uM, 1 1.9 uCi/mmol). ABA and its metabolites were extracted and analyzed as described in "Materials and Methods." Aliquots of the samples were counted separately to determine total uptake. The percentage of each metabolite was calculated and used to determine the absolute amount of ['4C]ABA and its metabolites. The molarities of ['4C]ABA and its metabolites were calculated based on the radioactivity in each peak and the estimation that 10 aleurone layers contain 0.1 ml of H20. Hours of Incubation with ['4C]ABA 2 % of ['4C]ABA in the medium taken up by 10 aleurone layers Mol ['4C]ABA from the medium taken up by 10 aleurone layers % of radioactivity in cells remaining as ABA M concn. of ['4C]ABA in aleurone layers 60
I
~ |
12
4
15.2
22.8
1.2 x 10-9
70.6
35.8
1.9 x 10-9
44.4
3.0 x 10-9
59.5
9.0 x 106
24
3.7 x 10-9
14.6
9.5
4.4 x 106
3.5 x 106
1. x 10-5
B. PA
6C N 40
(i 4C ,2C,
;t
8
20
A. ABA -
l-
';
60
C. DPA _
D.
Conjugotes and metabolites ~~~~~~~~~polar
A. ABA C. DPA
2C)
40
'4-
8
12
4 16 20 8 Hour of Preincubation
12
16
20
FIG. 4. The effects of length of preincubation with ABA on the metabolism of [3H]ABA in barley aleurone layers. Barley aleurone layers were preincubated in succinate buffer with or without 10-s M ABA for various lengths of time. The tissue was then labeled in I ml succinate buffer with I MCi [3H]ABA for 2 h. ABA and its metabolites were extracted and analyzed as described under "Materials and Methods." The percentage of total radioactivity in individual peaks on TLC plates was plotted versus the preincubation time. Control (@-@); ABA preincubation sample (O---4). 4
Q4
3
M QF -4
Li
a\ a\
2-
I
0
4
8
12
16
20
Hour of Preincubotion FIG. 5. Effect of preincubation with ABA on the enhancement of PA formation. Similar to what was described under Figure 4 except that the results from Figure 4B and other similar experiments were graphed as the ratio of per cent PA in the ABA-treated tissue to that in the control (% PA (+ABA)/% PA [control]) versus the length of preincubation.
O-D.
t Con,ugotes ond
polar
metobolites OD -7
4
_
10
10\10
20
-
5
CO log
-7
-5
[ABA] M
FIG. 6. Dosage-response curve of the self-induction of ABA metabolism in barley aleurone layers. Barley aleurone layers were preincubated in succinate buffer with various concentrations of ABA for 12 h. The tissue was then labeled in I ml succinate buffer with I gCi [3H]ABA for 2 h. ABA and its metabolites were extracted and analyzed as described under "Materials and Methods." The percentage of total radioactivity in individual peaks on TLC plates was plotted versus the ABA concentrations during the preincubation.
much stronger effect on PA formation. Again, there was a general decrease in the amount of [3H]ABA when the formation of [3H]PA was enhanced by the pretreatment (Fig. 6A). The
gave a
relative amount of [3H]DPA remained low fluctuating between 0.5% and 3% of total radioactivity (Fig. 6C). The formation of conjugates and other polar metabolites was enhanced when the ABA concentrations varied from 10-8 to 10-' M with optimal concentration at 101 M (Fig. 6D). Effect of Pretreatment with PA on the Metabolism of [3Hj ABA. After having established that preincubation of barley aleurone layers with ABA enhances the conversion from [3H]ABA to [3H]PA, we considered whether or not exogenously applied PA could also affect ABA metabolism. To test this, aleurone layers were preincubated with 10-1 M ABA, 10-5 M PA, or buffer only for 12 h followed by labeling with [3H]ABA for 2 h. As shown in Figure 7, ABA preincubation caused approximately a 4-fold enhancement in the conversion from [3H]ABA to [3H]PA, yet it had no apparent effect on the formation of [3H]DPA. On the other hand, preincubation with PA had no effect on the formation of either [3H]PA or [3H]DPA. These results indicate that ABA, but not PA, can induce a change in ABA metabolism
1130
UKNES AND HO
Table II. Effect of Transcription and Translation Inhibitors on the Metabolism ofABA in Barley Aleurone Layers Barley aleurone layers were incubated under the various conditions indicated above for 4 h. The tissue was then labeled in fresh medium containing inhibitor and [3H]ABA (I #Ci/ml) for 2 h. ABA and its metabolites were extracted and analyzed as described under "Materials and Methods." Pretreatment Peak + 10-5 M
ABA
3.C4- A Control 2. 2.C
F
I.c
l.C a'.r
Plant Physiol. Vol. 75, 1984
conjugates and
polar metabolites
-ABA
3.0 )_B. +ABA_ 2.5 I conugate and PA ABA DPA 2.C 1.5 1.0
conjugates and
cpm (%
Control ABA PA DPA
Conjugates and other polar metabolites
PA
ABA PA DPA
Conjugates and other polar metabolites
C.P+PPAB
3814 (57.2) 1351 (20.3) 271 (4.1)
1746 (22.8)
877 (13.2)
5253 (80.0) 154 (2.3) 197 (3.0)
5329 (83.3) 134 (2.1) 192 (3.0)
695 (10.6)
531 (8.3)
5304 (82.2) 75 (1.2) 174 (2.7)
5770 (84.4) 36 (0.5) 163 (2.4)
661 (10.2)
619 (9.1)
+ Cycloheximide (10 /Ig/
ml)
polar metabolites
-3
0
3
of total)
4540 (59.4) 613 (8.0) 329 (4.3)
+ Cordycepin (10' M)
2ko 2.5 2.0 i.5 1.0 Q5
ABA
6
9
12
cm
FIG. 7. Effect of pretreatment of barley aleurone layers with PA on the metabolism of [3H]ABA. Barley aleurone layers were preincubated in succinate buffer with or without either 10-s M ABA or 10' M PA for 12 h. After incubation fresh medium containing I ACi [3H]ABA/ml was added and the tissue was incubated for 2 h. ABA and its metabolites were extracted and analyzed as described under "Materials and Methods." The radioactivity in TLC sections was graphed versus the distance travelled in cm.
in barley aleurone layers. Effect of Transcription and Translation Inhibitors on ABA Metabolism. As a preliminary attempt to investigate the regulation of ABA metabolism in barley aleurone layers, we have studied whether the formation of ABA metabolites is affected by inhibitors of protein (cycloheximide) or RNA (cordycepin) synthesis. In the presence of either cycloheximide or cordycepin, less [3H]ABA was metabolized to PA, DPA and other compounds. Furthermore, the enhancement of [3H]PA formation induced by ABA pretreatment failed to take place in the presence of these inhibitors (Table II). Tissue Specificity of the Self-Inducted ABA Metabolism. Is the apparent self-induction of ABA conversion to PA unique to barley aleurone layers? To study the tissue specificity of this phenomenon, we have examined ABA metabolism in many different tissues as summarized in Table III. Besides barley aleurone layers only wheat aleurone layers showed an enhanced PA formation upon preincubation with ABA. All the other tissues, including leaf disc of barley and soybean, corn root tips, and barley embryos, do not possess this character. DISCUSSION In this work we have demonstrated that the metabolism of [3HJABA to [3H]PA is enhanced in barley aleurone layers when the tissue is pretreated with ABA. On the other hand, pretreat-
ABA PA DPA Conjugates and other polar metabolites
ment with ABA or PA has no effect on the conversion of [3H] PA to the next metabolite, [3H]DPA. Our observations indicate that ABA specifically enhances the rate of the biochemical step converting ABA into PA. The other evidence supporting this notion is summarized below. (a) When the amount of [3H]PA is increased in ABA-pretreated tissue, there is always a concomitant decrease in [3H]ABA. (b) Sufficiently short labeling times (2-4 h) were used to study the metabolism of [3H]ABA. Therefore, it is likely that our TLC observations reflect the rate of formation, rather than the accumulation, of the ABA metabolites. (c) The increase of [3H]PA is not due to the increase of uptake of [3H]ABA, which is not affected by ABA pretreatment. (d) Since [3H]DPA formation is not affected by either ABA or PA, the increase of [3H]PA in ABA-pretreated tissue is not likely to be the consequence of a simple mass action due to the presence of a large amount of ABA. The apparent enhancement of [3H]PA formation in ABApretreated tissue is 2- to 5-fold over the control (pretreated with buffer only). However, this appears to be an underestimation due to the changes in pool size of ABA. Our calculations, based on published data of Goldbach and Michael (6), indicate that the concentration of endogenous ABA in barley aleurone layers is about 4 x I0- M. However, during the ABA pretreatment the endogenous ABA concentration has increased to about 3 x I0O to IO-' M (Table I). This reflects at least a 10-fold increase in ABA pool size which causes a decrease in specific radioactivity of [3H]ABA in the ABA pretreated tissue. Taking this factor into consideration, the actual enhancement of [3H]ABA metabolism in the ABA-pretreated sample is at least 10 times higher than what we have observed, i.e. there should be 20- to 50-fold more ABA being converted to PA in the ABA-pretreated sample than that in the control. Since the endogenous ABA concentration in the control is
SELF-INDUCTION OF ABA CONVERSION TO PA
1 131
Table III. A Survey ofABA Metabolism in Various Plant Tissues Barley leaf discs, soybean leaf discs, and corn root tips were incubated for 4 h to recover from excision injury and then pretreated with and without (control) 10-s M ABA for 6 h before being labeled with 33H]ABA for 2 h. Wheat aleurone layers were preincubated for 24 h with and without (control) 1O-s M ABA and then labeled for 2 h with [3H]ABA. Half-d-old barley embryos and 1.5-d-old barley embryos were grown with or without (control) 10-5 M ABA and then labeled for 3 h with [3H]ABA. For all the above tissues, I ACi of [3H] ABA/ml was used for labeling. Distribution of Radioactivity Pretreatment Tissue Conjugates and other DPA ABA PA polar metabolites % of total radioactivity 15.7 3.8 48.3 31.3 Control Barley leaf disc 18.4 4.2 + ABA 30.9 45.9 1.7 11.4 5.3 Control 79.0 Soybean leaf disc + ABA 1.1 14.8 3.5 80.3 49.0 28.9 16.9 Control 4.3 Corn root tip + ABA 52.6 21.3 21.5 4.3 12.5 8.5 12.9 Wheat aleurone layers Control 63.5 + ABA 11.6 18.2 45.6 23.0 Control 45.7 29.1 10.0 12.8 Barley embryos (0.5 d old) 6.1 10.5 + ABA 26.3 54.3 25.3 36.3 11.6 22.9 Control Barley embryos (1.5 d old) + ABA 6.7 34.8 13.4 43.5
already 4 x 10-7 M, it is not surprising to see that the ABA concentration during the pretreatment has to be I0O M or higher to significantly enhance the formation of PA (Fig. 6). Is the enhancement of PA formation a means for the tissue to scavenge the excessive amounts of ABA? A similar scavenging mechanism involving IAA metabolism has been observed (14). The activity of IAA oxidase, an enzyme converting IAA to inactive metabolites, is enhanced in IAA-treated tissues. However, this is unlikely in our case because isolated PA has been shown to be biologically active in barley aleurone layers (3, 9). To get rid of the excessive ABA, the tissue would have to convert ABA to at least DPA which possesses little or no biological activity. Actually, the most efficient way to temporarily remove surplus ABA is probably the formation of conjugates. We have observed that the formation of conjugates and polar ABA metabolites is slightly enhanced under certain circumstances. However, since TLC was used in this work we were unable to resolve these compounds in an unequivocal fashion. The more powerful HPLC technique will be necessary for this purpose. The ABA induction of PA formation could be regarded as a typical substrate induction case in which the substrate induces the enzyme for its own metabolism. The best known substrate induction example in higher plants is nitrate reductase induction by nitrate. In this case, nitrate induces its own metabolism via the synthesis of nitrate reductase (and nitrite reductase) in order to be converted to amonium which can then be used to form amino acids and other nitrogen containing compounds. We have observed analogous situations in the metabolism of ABA, i.e. ABA induces its own metabolism to PA. It has been reported that ABA is able to induce several proteins in barley aleurone layers (8, 9, 11, 15). The time course and the dosage-response curve of the induction of new proteins correlate very well with those of the self-induction of ABA metabolism (Ho, manuscript in preparation). In addition, the self-induction of ABA metabolism can be inhibited by transcription (cordycepin) and translation (cycloheximide) inhibitors, indicating that the formation of new proteins is necessary for this process. Therefore, it is likely that some of the ABA-induced proteins are involved in the metabolic conversion from ABA to PA. Considering the fact that PA is as biologically active as ABA in barley aleurone layers, we propose that PA is the active component in this tissue. When
aleurone layers are treated with ABA, it actively converts itself into PA via the induction of new proteins. Phaseic acid will then exert a regulatory role in the synthesis of a-amylase and other related processes. Experiments are now underway to test whether ABA indeed induces the specific enzyme, a mono-oxygenase, which converts ABA into PA. It should be noted, however, that the self-induction of ABA metabolism only takes place in two of the plant tissues that we have examined, i.e. the wheat and barley aleurone layers. Therefore, it could be a process unique to these particular tissues. This is in line with the observations that, despite its effectiveness in barley aleurone layers, PA is not very active in many other tissues (for review, see 17). Nonetheless, our observations in this work warrant further investigation into the potential physiological roles of PA in germination related processes. Acknowledgments-We are grateful to Dr. Thomas D. Sharkey for the generous gifts of highly purified PA and DPA, and to Rachel W. Hammerton for introducing us to the preadsorbent TLC system. We also thank Jose Segura for scraping numerous TLC plates, and Sheila Hunt for the prepartion ofthis manuscript.
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