Planta
Planta 150, 312-320 (1980)
9 by Springer-Verlag 1980
A Study of Abscisic Acid Uptake by Apical and Proximal Root Segments of Phaseolus coccineus L. Mary C. Astle and Philip H. Rubery University of Cambridge, Department of Biochemistry, Tennis Court Road, Cambridge, CB2 IQW U.K.
Abstract. 1. We investigated the pH and concentration dependence of abscisic acid uptake by short segments taken from different zones along the length of primary roots of Phaseolus coccineus L. (Runner bean). Tissue from all regions studied, up to and including the zone of lateral root initiation showed a non-saturable uptake component identifiable with passive diffusion of the undissociated species of abscisic acid. The net uptake increased through the elongation zone towards the apex, perhaps principally due to the increasing relative volume of cytoplasm (pH value 7-8 ; cf pH 4-6 for vacuole) acting as an anion trap. A saturable uptake component, Km=2.6+0.8 gmol dm 3, is restricted to the apical 4-6 mm of the root (including lateral roots), is not of metabolic origin, and is likely to be a carrier. 2. No polarity of transport could be detected using donor blocks containing [2-14C]abscisic acid applied to 15 mm or 40 mm segments whose apical 10 mm had been removed; if the elongation zone were present in the test segments, a distribution of radioactivity that might be expected from acropetal polarity was obtained, but which may simply be accounted for by the greater uptake capacity of the elongating, relatively unvacuolated cells in the extending region of the root. Key words: Abscisic acid Carrier (ABA) - Geotropism - Phaseolus - Roots - Transport.
Introduction Abscisic acid transport has been comparatively little investigated although it is likely to be an important factor influencing hormone concentration at sites of action. In intact seedlings, exogenous ABA migrates Abbreviations: ABA=abscisic acid; IAA =indol-3-yl acetic acid
0032-0935/80/0150/0312/$01.80
from leaves to growth points (eg Shindy et al. 1973), but no coherent picture has yet emerged regarding polar ABA transport. Basipetal polar transport has been found in Coleus only in young stem tissue and petioles (D6rffling and B6ttger 1968; Veen 1975) and no polarity was observed in cotyledonary petioles of Gossypium hirsutum (Ingersoll and Smith 1971). The most comprehensive study has been made using Phaseolus coccineus (runner bean, synonym multiflorus) roots. It was concluded that ABA transport by excised segments of primary roots (apical 2 mm discarded) was acropetally polarised (Hartung and Behl 1974). Acropetal movement required living cells and was decreased by treatments which perturb normal energy metabolism, as was basipetal movement (Hartung 1975). Using 5 cm segments of mature tissue (CUt 1.5 or 2.5 cm behind the apex), acropetal transport was found to be located in the stele, with some radial movement to the cortex (Hartung and Behl 1975). There is also evidence (mostly indirect) which suggests that ABA can move laterally in the caps of geotropically stimulated roots, and basally to the elongation zone where it is proposed differentially to inhibit growth on the lower side of the root (Wilkins 1977; Pilet 1975, 1978). None of these studies considered the fundamental problem of how ABA actually crosses membranes and enters cells. Yet a clear understanding of transport polarity and also the role of ABA in geotropism will require such knowledge. ABA is a low molecular weight lipophilic weak acid (pK=4.5); experiments using suspension cultured Parthenocissus tricuspidata crown gall cells (Rubery, unpublished) suggest that it shares the transport features of IAA, other auxins, and benzoic acid that depend on this physicochemical property (Rubery and Sheldrake 1973, 1974; Rubery 1977). Exemplified by IAA, the transmembrane transport is dominated by diffusion of the protonated form (IAAH) which has high lipid solubility relative to
M.C. Astle and P.H. Rubery: ABA Uptake by Root Segments I A A - . T h e a p p r o a c h to p a s s i v e flux e q u i l i b r i u m t h u s reflects m a i n l y e q u i l i b r a t i o n o f I A A H w i t h t h e c o n s e q u e n c e t h a t r e l a t i v e l y a l k a l i n e c o m p a r t m e n t s (eg c y t o p l a s m ; p H 7 8) will act as ' a n i o n t r a p s ' a n d a c c u m u l a t e a u x i n f r o m m o r e acidic e n v i r o n m e n t s . O t h e r indications that an anion trap process may contribute to A B A t r a n s p o r t a r e t h a t A B A d i s t r i b u t i o n b e t w e e n i s o l a t e d Spinacea c h l o r o p l a s t s a n d i n c u b a t i o n m e d i u m c a n be a c c o u n t e d for by p e r m e a t i o n o f A B A H , as c a n A B A c o m p a r t m e n t a t i o n in l e a f cells ( H e i l m a n n et al. 1980). A l s o A B A will p a r t i t i o n i n t o lipid b i l a y e r membranes, opening ion transport channels (Lea and C o l l i n s 1979). T h e r e a d y u p t a k e a n d efflux o f A B A by Cornmelina l e a f e p i d e r m a l strips, w i t h a c c u m u l a t i o n by t h e s t o m a t a l c o m p l e x ( W e y e r s a n d H i l l m a n 1979 a, b), c o u l d reflect p r i n c i p a l l y A B A H m o v e m e n t . The 'chemiosmotic model' proposed for polar a u x i n t r a n s p o r t ( R u b e r y a n d S h e l d r a k e 1974; R a v e n 1975; G o l d s m i t h 1977) is in p r i n c i p l e g e n e r a l l y a p p l i c a b l e to s u f f i c i e n t l y p e r m e a b l e l i p o p h i l i c w e a k a c i d s p r o v i d e d t h a t a s u i t a b l e p l a s m a m e m b r a n e c a r r i e r be a s y m m e t r i c a l l y l o c a t e d at o n e e n d o f t h e cells in t h e t r a n s p o r t p a t h w a y in o r d e r to b r i n g a b o u t a g r e a t e r l o c a l p e r m e a b i l i t y to the a c i d ' s a n i o n r e l a t i v e to its p r o t o n a t e d species ( R u b e r y 1977, 1979). S i n c e p o l a r A B A t r a n s p o r t h a d b e e n r e p o r t e d in r u n n e r b e a n r o o t s ( H a r t u n g a n d B e h l 1974, I 9 7 5 ; H a r t u n g 1975), we u s e d this tissue for a n initial s t u d y o f the m e c h a n i s m s c o n t r i b u t i n g to t r a n s m e m b r a n e A B A m o v e m e n t in s e g m e n t s f r o m v a r i o u s r e g i o n s o f the r o o t , i n c l u d i n g the g e o t r o p i c a l l y r e a c t i v e a p i c a l z o n e . E v i d e n c e was o b t a i n e d t h a t n o n s a t u r a b l e d i f f u s i v e u p t a k e o f A B A H o c c u r r e d in all r e g i o n s i n v e s t i g a t e d , whereas a saturable carrier-mediated component of u p t a k e w a s d e t e c t e d o n l y f r o m t h e r o o t tip to the end o f t h e e l o n g a t i o n z o n e . P o l a r A B A t r a n s p o r t d i d n o t o c c u r in r e g i o n s o f t h e r o o t t h a t h a d c e a s e d to e l o n g a t e a n d c o u l d n o t be u n a m b i g u o u s l y d e m o n s t r a t e d in e l o n g a t i n g tissue, w h e r e i n t e r p r e t a t i o n w a s c o m p l i c a t e d b y a g r a d i e n t o f i n c r e a s i n g A B A net u p take capacity associated with the progressively greater p r o p o r t i o n o f cell v o l u m e o c c u p i e d by c y t o p l a s m tow a r d s t h e r o o t tip.
313 to germination at 25~ between damp absorbent paper towels (2 5 d). For experiments with donor blocks that required long straight roots, soaked seeds were pinned, between moist paper towels, to boards which were placed vertically in an enclosed dark tank and kept at 25 ~ C in darkness. Seedlings that were abnormally grown, fungally contaminated, or in which the plumule was emerging were discarded. Experiments repeated at different seasons gave similar results, Transverse segments of the desired length were either cut from the entire root up to the region of lateral growth (designated pooled segments) or were cut from specific root regions and designated as follows: 'subapical segments' taken from i 2 mm behind the root apex; 'root hair segments' taken from the root hair region starting about 12 mm behind the tip. Except where otherwise noted, the apical 1 2 mm of the root (designated 'root tips') was discarded. The segments were kept for 30-60 min in ice-cold sucrose (I.3% w/v) prior to use in uptake experiments. Separation into stele and cortex at the endodermis (verified by phloroglucinol staining) was achieved mechanically (Hartung and Beh11975) using 5 6 cm long roots with the apical 1 cm discarded. Outer cylinders were longitudinally bisected, to expose alI surfaces to the incubation medium before cutting to the desired length.
Incubation oJ Exeised Segments The procedure was based on that of Davies and Rubery (1978). In summary, 7 to 20 randomised segments (2 5 mm, of specified length) were shaken at 25~ in boiling tubes containing 3 cm 3 incubation medium (I.3% w/v sucrose buffered at the desired pH with McIlvaine's Na phosphate/citrate buffer, plus other additives as described in context including radioactive ABA or benzoic acid). In certain experiments the amount of [14C]ABA added was adjusted to reflect the differing specific activities of batches obtained from Amersham. The pH range covered was from pH 3.5 (10.1 mmol dm -3 Na2HPO4+I1.6 mmol dm -3 citric acid) to pH 7.0 (27.5 mmol d m a Na2HPO4+2. 9 mmol dm -3 citric acid), Segments were harvested to terminate uptake by rapid filtration under reduced pressure and washed briefly with 2 cm 3 ice-cold 1.3% (w/v) sucrose. (The washing procedure was found to improve the reproducibility of the results). The segments were maintained under suction for a further 15 s after the bulk of the wash had drained, and were then weighed and placed in a vial containing 6 cm 3 aqueous scintillation fluid (toluene/Triton X- 100/H20, 20 : 10 : 3 by vol containing 2.12 g d m -3 2,5-diphenyloxazole). Samples were counted after 12 h when extraction of radioactivity was complete and corrected for quenching by automatic external standard channels ratio. Results were corrected for remaining extracellular radioactivity using a zero-time incubation. In experiments with 14 mm long segments, incubations were performed in small conical flasks using 18 cm 3 incubation medium. The basal ends of the segments had been marked with Rotring china tinta ink, and on termination of incubation, aligned groups of segments were cut into 2 mm slices, successively from either end. The seven groups of slices obtained were weighed and counted separately.
Use of Agar Donor Blocks
Materials and Methods DL-(cis, trans)[2-14C]ABA (348-418 GBq mol a stored in darkness at 2~ C as a 0.3 mmol dm-3 ethanolic solution) and [1-1r acid (2.14 TBq mol- 1 stored as a frozen aqueous solution of the K salt - Rubery 1977) were obtained from the Radiochemical Centre, Amersham. A racemic mixture of non-radioactive ABA was obtained from Sigma.
Growth of Seedlings and Manipulation of Root Tissue Runner bean (Phaseolus coccineus L. cv Prizewinner) seeds were obtained locally and soaked in cold running tap water (3 h) prior
Segments (6, 15 or 40 mm tong) were ringed with lanolin and placed horizontally on glass microscope slides over damp absorbent paper in enclosed Petri dishes (10 segments per dish), with their morphologically apical or basal ends in close contact with cylindrical agar donor blocks. The blocks (3 mm long, 3 mm diameter; 1% Agar Noble adjusted to about pH 5.4 with Na citrate/citric acid buffer) incorporated 0.5 ~tmol dm -3 or 1.0btmol dm -3 [2t4C]ABA, added while the agar was just molten. Similar ABA-free receiver blocks were used in some experiments. After incubation in darkness at 25 ~ C for 12 h, the aligned segments were cut successively into 2, 3 or 5 mm sIices, according to the starting length used and counted after extraction into aqueous scintillation fluid.
314
M.C. Astle and P.H. Rubery: ABA Uptake by Root Segments
Extraction of Radioactivity from Segments and Thin Layer Chromatography On ~termination of the incubation, segments were extracted twice in 10 cm 3 methanol/CO2(s) (freezing conditions maintained for 30 rain), and finally filtered when at room temperature. About 95% of the tissue radioactivity was extracted. The methanolic extracts were reduced to dryness by evaporation under reduced pressure at 35~ in dim light and redissolved in 0.5 cm3 methanol for chromatography using either 0.25 mm silica gel or 0.1 mm cellulose layers on plastic sheets (Macherey-Nagel). The plates were prewashed in ethanol/acetic acid (98:2 by vol). Internal standards of radioactive ABA and benzoic acid were also taken through the extraction procedure for chromatographic analysis. The solvent systems used for ABA with silica plates were benzene/acetic acid (50:20 by vol, Rf=0.32); propan-l-ol/ethyl acetate/HzO (4:5:2 by vol, Rf = 0.58); and toluene/ethylacetate/acetic acid (40:5:2 by vol, Rf=0.18 after three successive developments to 17 cm past the origin). The solvents for benzoic acid were benzene/methanol/ acetic acid (90:16:8 by vol, Rf=0.5 on silica gel) and butan-l-ol/ ethanol/3 mol dm-3,ammonia aq (4:1:5 by vol, Rf=0.61 on cellulose). All solvents were redistilled. After development~ the plates were dissected into 2.5.0.5 cm strips and put into small vials containing 2 cm 3 scintillant (3.5 g dm -3 2,5-diphenyloxazole in toluene) for counting. The positions of ABA and benzoic acid were determined using radioactive markers.
The Dissociation Constant of ABA The pK of ABA was determined spectrophotometrically as about 4.5 (cf. Lea and Collins 1979). The pH dependence of its absorbance (Cornforth and Milborrow 1966) at 262 nm (maximum in acid solution) and at 245 nm (maximum in alkaline solution) was measured by adding 3 tool dm -3 HC1 dropwise to 67 gmol dm 3 sodium ABA. There was an isosbestic point at 258 rim. It was assumed that absorbance changes reflect protonation of the carboxyl group.
Results and Discussion The initial c h a r a c t e r i s a t i o n o f the b r o a d features o f A B A u p t a k e b y b e a n r o o t tissue was c a r r i e d o u t using p o o l e d segments. Subsequently, the r e s o l u t i o n o f the analysis was increased using segments cut f r o m particular r o o t zones.
Time Course and p H Dependence of [2-14C]ABA Uptake by Pooled Root Segments T h e u p t a k e o f [2-14C]ABA (1.0 g m o l d m - 3) by 4 m m long segments was f o l l o w e d (0-60 rain) at p H 4.0, 5.0 a n d 6.5 (Fig. 1). T h e u p t a k e is increased as the p H b e c o m e s m o r e acidic, a n d is initially r a p i d b u t then slows to a steady rate, a t t a i n e d earlier at h i g h e r p H values. T h e first p h a s e p r o b a b l y m a i n l y represents u p t a k e into cells at the cut surfaces in direct c o n t a c t with the i n c u b a t i o n m e d i u m . T h e p r o g r e s s i n g s t e a d y u p t a k e , which continues for at least a further h o u r at p H 5.0 will reflect c o n t i n u i n g p e n e t r a t i o n o f A B A to cells within the segment a n d also any m e t a b o l i c conversions. A B A is slowly m e t a b o l i s e d by r u n n e r b e a n r o o t tissue: in the ' W e i s s e r R i e s e ' variety used
1"0
f O'8
~
9
g0-~
2e~
0
0
I
I 20
I 30 Time / mi~
I
40
I
50
I 60
Fig. 1. Uptake of [2-1~C]ABA (1 Ilmol dm -3) into 4 mm pooled root segments (9 per incubation) from 3 d-old seedlings over 0 t o 6 0 m i n . - A - p H 4 . 0 ; - o - p H 5 . 0 ; - m pH6.5
by H a r t u n g a n d Behl (1974, 1975), m e t a b o l i s m o n l y b e c a m e significant after 11 h e x p o s u r e to r a d i o a c t i v e h o r m o n e , a l t h o u g h in our e x p e r i m e n t s a b o u t 2 0 % A B A was c o n v e r t e d to a less p o l a r substance after 2 h i n c u b a t i o n (Table 1). In s u b s e q u e n t experiments, i n c u b a t i o n times were generally limited to 5 min, to a p p r o x i m a t e to the initial rate o f u p t a k e , a n d when no d e t e c t a b l e m e t a b o l i s m o f A B A h a d o c c u r r e d (Table 1). A B A u p t a k e (5 min, 0.5 g m o l d m - 3) shows a sigm o i d a l d e p e n d e n c e on external p H (Fig. 2) over the range p H 3.5 to p H 7.0, with a m i d p o i n t at a b o u t p H 4.6, close to the p K o f A B A . Such b e h a v i o u r has p r e v i o u s l y been f o u n d for l i p o p h i l i c w e a k acids using c u l t u r e d cells (including A B A , R u b e r y u n p u b lished) a n d stem tissues ( R u b e r y a n d S h e l d r a k e i973; R u b e r y 1977; Davies a n d R u b e r y 1978) a n d is consistent with passive A B A H p e r m e a t i o n being a m a j o r c o m p o n e n t o f net u p t a k e . This i n t e r p r e t a t i o n is supp o r t e d b y the ability o f 2 , 4 - d i n i t r o p h e n o l a n d c a r b o n y l c y a n i d e p - t r i f l u o r o m e t h o x y p h e n y l h y d r a z o n e (Fig. 3) to decrease net A B A u p t a k e in a m a n n e r c o n s o n a n t with their a c t i o n as p r o t o n i o n o p h o r e s , t e n d i n g to equilibrate p H g r a d i e n t s across m e m b r a n e s . C o m p a r ison with rates of I A A u p t a k e b y r o o t segments (not shown) suggests t h a t I A A H is a b o u t 5 times m o r e p e r m e a n t t h a n A B A H . This is n o t u n e x p e c t e d since A B A is a larger a n d s o m e w h a t m o r e p o l a r m o l e c u l e
M.C. Astle and P.H. Rubery: ABA Uptake by Root Segments 1. Extent of metabolic conversions of [2-14C]ABA and[1-14C]benzoic acid by bean root segments. 4 mm segments were incubated at 25~ C in buffered (pH 5.0) uptake media; basal agar donor blocks were applied to 15 mm segments. Per-cent conversion to methanol-soluble radioactivity not identical with authentic markers after thin layer chromatography (see Materials and Methods) is shown Table
Root region incubated
Time
% 14C in metabolite
ABA
4 mm segments: sub-apical root hair region
Benzoic acid
5min 2h
undetectable" 23b, 25 ~
10 89
5min 2h
undetectable" 17d
91
I5 mm segments with basal donor blocks : sub-apical 12 h root hair region 12 h
47 ~ 51e
100 _
0.5 [tmol dm -3 [1-14C]benzoate was supplied. [2-a4C]ABA concentrations: a, 5 gmol dm-3 to accommodate short uptake time; b, 0.5 gmol dm 3; c, 0.5 gmol dm-3+5 gmol dm -3 nonradioactive ABA; d, 1.0 gmol dm 3; e, 2.5 gmol dm -3 to accommodate slower uptake from agar blocks
A
100
70
60
5O & ~o ~o
< 10 0
0
1
100
1000
[Additive]/uM
Fig. 3. The effects of 2,4-dinitrophenol (0 1.000 gmol d m - 3) ( o - ) and carbonylcyanide p-trifluoromethoxy phenylhydrazone (020 gmol dm 3) ( [] ) on [2-14CIABA uptake (0.5 gmol dm -3, 2 h,
pH 5.0) into 3 mm sub-apical segments (12 per incubation) from 3 d-old seedlings
with a lower ether/water p a r t i t i o n coefficient t h a n I A A ( C o l l a n d e r 1949; M i l b o r r o w 1967).
Concentration Dependence of ABA Uptake by Pooled Root Segments
90
A B A u p t a k e (0 160 g m o l d m -3) after 5 rain i n c u b a tion was d e t e r m i n e d at p H 4.0, 5.0 a n d 6.5. There was n o i n d i c a t i o n of overall s a t u r a t i o n at a n y of these p H values (Fig. 4). However, a saturable comp o n e n t does a p p e a r to c o n t r i b u t e significantly at the lower c o n c e n t r a t i o n s where A B A u p t a k e is relatively higher. This is also shown in the inset of Fig. 4 as the effect of increasing c o n c e n t r a t i o n s of n o n - r a d i o a c tive A B A (0.5-6,0 g m o l d m - 3 ) t o i n h i b i t u p t a k e of 0.5 g m o l d m 3 [2_Ir A (5 min) at p H 5.0 to a c o n s t a n t p l a t e a u value, representing s a t u r a t i o n of a putative carrier. The u p t a k e c o m p o n e n t insensitive to u n l a b e l l e d A B A reflects c o n t i n u i n g n o n - m e d i a t e d diffusion of A B A H , or possibly the o p e r a t i o n of a low affinity carrier system.
80
70
60
E 50 S0 30 20 10 0
315
I
I
I
I
I
3
4
5
6
7
pH
Fig. 2. pH-dependence of [2-1~C]ABA uptake (0.5 gmol dm 3,
Characteristics of ABA Uptake by Different Regions of the Root
5 min) expressed as per cent of maximum uptake (found by extrapolation to about pH 3). 10-4 mm segments from 4 d-old seedlings were used per incubation. Maximum uptakes were for pooled segments (-o-) 4.2 dpm mg- 1 5 rain 1; for sub-apical segments ( 9 -) 8.6 dpm mg -1 5 min-l; for root hair region segments (_A_) 3.7dpmmg-1 5min 1
In order to investigate the u n i f o r m i t y of A B A u p t a k e characteristics of the different regions of the root collected in the pooled segments, batches of 4 m m segm e n t s were assembled from specified regions (see ma-
316
M.C. Astle and P.H. Rubery: ABA Uptake by Root Segments Table 2. Effect of a ten-fold excess of nonradioactive ABA on uptake of [2-14C]ABA (5 rain, pH 5.0) by segments from different regions of primary roots of 4 d-old seedlings (0.5 gmol dm -3 labelled ABA) and lateral roots of 5 d-old seedlings (0.8 gmol dm 3 labelled ABA)
v=
~
1.6
Significance was assessed using Student's t test
30 ~
Root region incubated
0
7-
0
2
without [IZC]ABA
with [12C]ABA
Terminal 2 mm, including cap.
2.85__0.15 (3)
2.30_+0.11 (4)
Subapical 4 mm, i-2 mm tip removed.
3.74_+0.11 (3)
2.57+0.03 (3) (p < 0,001)
4 mm segments, immediately before root hair region.
1.85_+0.04 (4)
1.81_+0.09 (4) (ns)
4 mm segments from root hair region.
1.37_+0.03 (3)
1.35_+0.10 (4) (ns)
2 mm segments from stele of root hair region.
6.76_+0.13 (3)
6.44+_0.34 (4) (ns)
2 mm segments from cortex of root hair region.
2.83Z0.13 (4)
2.62_+0.tl (4) (ns)
Terminal 3 ram, including cap.
4.05_+0.tl (4)
3.32_+0.07 (4) (p
i
-5
[IZc- ABA]
/"pM
I
t
5
10
Fig. 5. Dixon plot showing dependence of the saturable component of [2-~4C]ABA uptake (0.8 ~tmol dm -3, pH 5.0. 5 rain) on nonradioactive ABA concentration. 12.3mm sub-apical segments from 3 d-old seedlings were used per incubation. Magnitude of [2A4C]ABA uptake due to saturable component was determined by subtraction of diffusive component (plateau level of [2-1SC]ABA uptake at saturating non-radioactive ABA concentrations) from measured [2A4C]ABA uptake. Line drawn by least squares linear regression, using a weighting factor of v 3
levels in maize root apices (up to 0.25 gmol kg -1 fresh wt - Rivier et al. 1977) and Phaseolus vulgaris roots (0.12 gmol kg -1 fresh wt - Weiler 1980). Further investigations of the mechanism and driving forces of carrier-mediated ABA transport are in progress; one possibility is cotransport, as suggested previously for the auxin uptake carrier present in crown gall cells (Rubery 1978). Attempts to Detect Polar Transport of ABA in Bean Root Tissue In view of the restriction of the ABA carrier to the apical regions, we investigated the occurrence of polar ABA transport in longer segments of the primary roots, both including and excluding the apical tissue. An initial study involved incubating subapical 14 mm segments (first 2 mm discarded) for 2 h in 0.5 gmol dm -3 [2-14C]ABA at pH 5.0, followed by measurement of radioactivity present in sequential 2 mm slices. The resulting profile showed a strong gradient of radioactivity towards the apical end, such as might be expected if acropetal transport were occurring. However, division of the tissue into 2 mm slices before incubation with ABA revealed a comparable gradient in uptake capacity (Fig. 6 a). When the same protocol was followed with the next 14 mm long portion of the root ("root hair region", 16-30 mm from the apex), the radioactivity was symmetrically distributed, with increased uptake adjacent to both cut surfaces.
318
M.C. Astle and P.H. Rubery: ABA Uptake by Root Segments
z~0
?-0
0'6
~1'0
Planta 150, 312-320 (1980)
9 by Springer-Verlag 1980
A Study of Abscisic Acid Uptake by Apical and Proximal Root Segments of Phaseolus coccineus L. Mary C. Astle and Philip H. Rubery University of Cambridge, Department of Biochemistry, Tennis Court Road, Cambridge, CB2 IQW U.K.
Abstract. 1. We investigated the pH and concentration dependence of abscisic acid uptake by short segments taken from different zones along the length of primary roots of Phaseolus coccineus L. (Runner bean). Tissue from all regions studied, up to and including the zone of lateral root initiation showed a non-saturable uptake component identifiable with passive diffusion of the undissociated species of abscisic acid. The net uptake increased through the elongation zone towards the apex, perhaps principally due to the increasing relative volume of cytoplasm (pH value 7-8 ; cf pH 4-6 for vacuole) acting as an anion trap. A saturable uptake component, Km=2.6+0.8 gmol dm 3, is restricted to the apical 4-6 mm of the root (including lateral roots), is not of metabolic origin, and is likely to be a carrier. 2. No polarity of transport could be detected using donor blocks containing [2-14C]abscisic acid applied to 15 mm or 40 mm segments whose apical 10 mm had been removed; if the elongation zone were present in the test segments, a distribution of radioactivity that might be expected from acropetal polarity was obtained, but which may simply be accounted for by the greater uptake capacity of the elongating, relatively unvacuolated cells in the extending region of the root. Key words: Abscisic acid Carrier (ABA) - Geotropism - Phaseolus - Roots - Transport.
Introduction Abscisic acid transport has been comparatively little investigated although it is likely to be an important factor influencing hormone concentration at sites of action. In intact seedlings, exogenous ABA migrates Abbreviations: ABA=abscisic acid; IAA =indol-3-yl acetic acid
0032-0935/80/0150/0312/$01.80
from leaves to growth points (eg Shindy et al. 1973), but no coherent picture has yet emerged regarding polar ABA transport. Basipetal polar transport has been found in Coleus only in young stem tissue and petioles (D6rffling and B6ttger 1968; Veen 1975) and no polarity was observed in cotyledonary petioles of Gossypium hirsutum (Ingersoll and Smith 1971). The most comprehensive study has been made using Phaseolus coccineus (runner bean, synonym multiflorus) roots. It was concluded that ABA transport by excised segments of primary roots (apical 2 mm discarded) was acropetally polarised (Hartung and Behl 1974). Acropetal movement required living cells and was decreased by treatments which perturb normal energy metabolism, as was basipetal movement (Hartung 1975). Using 5 cm segments of mature tissue (CUt 1.5 or 2.5 cm behind the apex), acropetal transport was found to be located in the stele, with some radial movement to the cortex (Hartung and Behl 1975). There is also evidence (mostly indirect) which suggests that ABA can move laterally in the caps of geotropically stimulated roots, and basally to the elongation zone where it is proposed differentially to inhibit growth on the lower side of the root (Wilkins 1977; Pilet 1975, 1978). None of these studies considered the fundamental problem of how ABA actually crosses membranes and enters cells. Yet a clear understanding of transport polarity and also the role of ABA in geotropism will require such knowledge. ABA is a low molecular weight lipophilic weak acid (pK=4.5); experiments using suspension cultured Parthenocissus tricuspidata crown gall cells (Rubery, unpublished) suggest that it shares the transport features of IAA, other auxins, and benzoic acid that depend on this physicochemical property (Rubery and Sheldrake 1973, 1974; Rubery 1977). Exemplified by IAA, the transmembrane transport is dominated by diffusion of the protonated form (IAAH) which has high lipid solubility relative to
M.C. Astle and P.H. Rubery: ABA Uptake by Root Segments I A A - . T h e a p p r o a c h to p a s s i v e flux e q u i l i b r i u m t h u s reflects m a i n l y e q u i l i b r a t i o n o f I A A H w i t h t h e c o n s e q u e n c e t h a t r e l a t i v e l y a l k a l i n e c o m p a r t m e n t s (eg c y t o p l a s m ; p H 7 8) will act as ' a n i o n t r a p s ' a n d a c c u m u l a t e a u x i n f r o m m o r e acidic e n v i r o n m e n t s . O t h e r indications that an anion trap process may contribute to A B A t r a n s p o r t a r e t h a t A B A d i s t r i b u t i o n b e t w e e n i s o l a t e d Spinacea c h l o r o p l a s t s a n d i n c u b a t i o n m e d i u m c a n be a c c o u n t e d for by p e r m e a t i o n o f A B A H , as c a n A B A c o m p a r t m e n t a t i o n in l e a f cells ( H e i l m a n n et al. 1980). A l s o A B A will p a r t i t i o n i n t o lipid b i l a y e r membranes, opening ion transport channels (Lea and C o l l i n s 1979). T h e r e a d y u p t a k e a n d efflux o f A B A by Cornmelina l e a f e p i d e r m a l strips, w i t h a c c u m u l a t i o n by t h e s t o m a t a l c o m p l e x ( W e y e r s a n d H i l l m a n 1979 a, b), c o u l d reflect p r i n c i p a l l y A B A H m o v e m e n t . The 'chemiosmotic model' proposed for polar a u x i n t r a n s p o r t ( R u b e r y a n d S h e l d r a k e 1974; R a v e n 1975; G o l d s m i t h 1977) is in p r i n c i p l e g e n e r a l l y a p p l i c a b l e to s u f f i c i e n t l y p e r m e a b l e l i p o p h i l i c w e a k a c i d s p r o v i d e d t h a t a s u i t a b l e p l a s m a m e m b r a n e c a r r i e r be a s y m m e t r i c a l l y l o c a t e d at o n e e n d o f t h e cells in t h e t r a n s p o r t p a t h w a y in o r d e r to b r i n g a b o u t a g r e a t e r l o c a l p e r m e a b i l i t y to the a c i d ' s a n i o n r e l a t i v e to its p r o t o n a t e d species ( R u b e r y 1977, 1979). S i n c e p o l a r A B A t r a n s p o r t h a d b e e n r e p o r t e d in r u n n e r b e a n r o o t s ( H a r t u n g a n d B e h l 1974, I 9 7 5 ; H a r t u n g 1975), we u s e d this tissue for a n initial s t u d y o f the m e c h a n i s m s c o n t r i b u t i n g to t r a n s m e m b r a n e A B A m o v e m e n t in s e g m e n t s f r o m v a r i o u s r e g i o n s o f the r o o t , i n c l u d i n g the g e o t r o p i c a l l y r e a c t i v e a p i c a l z o n e . E v i d e n c e was o b t a i n e d t h a t n o n s a t u r a b l e d i f f u s i v e u p t a k e o f A B A H o c c u r r e d in all r e g i o n s i n v e s t i g a t e d , whereas a saturable carrier-mediated component of u p t a k e w a s d e t e c t e d o n l y f r o m t h e r o o t tip to the end o f t h e e l o n g a t i o n z o n e . P o l a r A B A t r a n s p o r t d i d n o t o c c u r in r e g i o n s o f t h e r o o t t h a t h a d c e a s e d to e l o n g a t e a n d c o u l d n o t be u n a m b i g u o u s l y d e m o n s t r a t e d in e l o n g a t i n g tissue, w h e r e i n t e r p r e t a t i o n w a s c o m p l i c a t e d b y a g r a d i e n t o f i n c r e a s i n g A B A net u p take capacity associated with the progressively greater p r o p o r t i o n o f cell v o l u m e o c c u p i e d by c y t o p l a s m tow a r d s t h e r o o t tip.
313 to germination at 25~ between damp absorbent paper towels (2 5 d). For experiments with donor blocks that required long straight roots, soaked seeds were pinned, between moist paper towels, to boards which were placed vertically in an enclosed dark tank and kept at 25 ~ C in darkness. Seedlings that were abnormally grown, fungally contaminated, or in which the plumule was emerging were discarded. Experiments repeated at different seasons gave similar results, Transverse segments of the desired length were either cut from the entire root up to the region of lateral growth (designated pooled segments) or were cut from specific root regions and designated as follows: 'subapical segments' taken from i 2 mm behind the root apex; 'root hair segments' taken from the root hair region starting about 12 mm behind the tip. Except where otherwise noted, the apical 1 2 mm of the root (designated 'root tips') was discarded. The segments were kept for 30-60 min in ice-cold sucrose (I.3% w/v) prior to use in uptake experiments. Separation into stele and cortex at the endodermis (verified by phloroglucinol staining) was achieved mechanically (Hartung and Beh11975) using 5 6 cm long roots with the apical 1 cm discarded. Outer cylinders were longitudinally bisected, to expose alI surfaces to the incubation medium before cutting to the desired length.
Incubation oJ Exeised Segments The procedure was based on that of Davies and Rubery (1978). In summary, 7 to 20 randomised segments (2 5 mm, of specified length) were shaken at 25~ in boiling tubes containing 3 cm 3 incubation medium (I.3% w/v sucrose buffered at the desired pH with McIlvaine's Na phosphate/citrate buffer, plus other additives as described in context including radioactive ABA or benzoic acid). In certain experiments the amount of [14C]ABA added was adjusted to reflect the differing specific activities of batches obtained from Amersham. The pH range covered was from pH 3.5 (10.1 mmol dm -3 Na2HPO4+I1.6 mmol dm -3 citric acid) to pH 7.0 (27.5 mmol d m a Na2HPO4+2. 9 mmol dm -3 citric acid), Segments were harvested to terminate uptake by rapid filtration under reduced pressure and washed briefly with 2 cm 3 ice-cold 1.3% (w/v) sucrose. (The washing procedure was found to improve the reproducibility of the results). The segments were maintained under suction for a further 15 s after the bulk of the wash had drained, and were then weighed and placed in a vial containing 6 cm 3 aqueous scintillation fluid (toluene/Triton X- 100/H20, 20 : 10 : 3 by vol containing 2.12 g d m -3 2,5-diphenyloxazole). Samples were counted after 12 h when extraction of radioactivity was complete and corrected for quenching by automatic external standard channels ratio. Results were corrected for remaining extracellular radioactivity using a zero-time incubation. In experiments with 14 mm long segments, incubations were performed in small conical flasks using 18 cm 3 incubation medium. The basal ends of the segments had been marked with Rotring china tinta ink, and on termination of incubation, aligned groups of segments were cut into 2 mm slices, successively from either end. The seven groups of slices obtained were weighed and counted separately.
Use of Agar Donor Blocks
Materials and Methods DL-(cis, trans)[2-14C]ABA (348-418 GBq mol a stored in darkness at 2~ C as a 0.3 mmol dm-3 ethanolic solution) and [1-1r acid (2.14 TBq mol- 1 stored as a frozen aqueous solution of the K salt - Rubery 1977) were obtained from the Radiochemical Centre, Amersham. A racemic mixture of non-radioactive ABA was obtained from Sigma.
Growth of Seedlings and Manipulation of Root Tissue Runner bean (Phaseolus coccineus L. cv Prizewinner) seeds were obtained locally and soaked in cold running tap water (3 h) prior
Segments (6, 15 or 40 mm tong) were ringed with lanolin and placed horizontally on glass microscope slides over damp absorbent paper in enclosed Petri dishes (10 segments per dish), with their morphologically apical or basal ends in close contact with cylindrical agar donor blocks. The blocks (3 mm long, 3 mm diameter; 1% Agar Noble adjusted to about pH 5.4 with Na citrate/citric acid buffer) incorporated 0.5 ~tmol dm -3 or 1.0btmol dm -3 [2t4C]ABA, added while the agar was just molten. Similar ABA-free receiver blocks were used in some experiments. After incubation in darkness at 25 ~ C for 12 h, the aligned segments were cut successively into 2, 3 or 5 mm sIices, according to the starting length used and counted after extraction into aqueous scintillation fluid.
314
M.C. Astle and P.H. Rubery: ABA Uptake by Root Segments
Extraction of Radioactivity from Segments and Thin Layer Chromatography On ~termination of the incubation, segments were extracted twice in 10 cm 3 methanol/CO2(s) (freezing conditions maintained for 30 rain), and finally filtered when at room temperature. About 95% of the tissue radioactivity was extracted. The methanolic extracts were reduced to dryness by evaporation under reduced pressure at 35~ in dim light and redissolved in 0.5 cm3 methanol for chromatography using either 0.25 mm silica gel or 0.1 mm cellulose layers on plastic sheets (Macherey-Nagel). The plates were prewashed in ethanol/acetic acid (98:2 by vol). Internal standards of radioactive ABA and benzoic acid were also taken through the extraction procedure for chromatographic analysis. The solvent systems used for ABA with silica plates were benzene/acetic acid (50:20 by vol, Rf=0.32); propan-l-ol/ethyl acetate/HzO (4:5:2 by vol, Rf = 0.58); and toluene/ethylacetate/acetic acid (40:5:2 by vol, Rf=0.18 after three successive developments to 17 cm past the origin). The solvents for benzoic acid were benzene/methanol/ acetic acid (90:16:8 by vol, Rf=0.5 on silica gel) and butan-l-ol/ ethanol/3 mol dm-3,ammonia aq (4:1:5 by vol, Rf=0.61 on cellulose). All solvents were redistilled. After development~ the plates were dissected into 2.5.0.5 cm strips and put into small vials containing 2 cm 3 scintillant (3.5 g dm -3 2,5-diphenyloxazole in toluene) for counting. The positions of ABA and benzoic acid were determined using radioactive markers.
The Dissociation Constant of ABA The pK of ABA was determined spectrophotometrically as about 4.5 (cf. Lea and Collins 1979). The pH dependence of its absorbance (Cornforth and Milborrow 1966) at 262 nm (maximum in acid solution) and at 245 nm (maximum in alkaline solution) was measured by adding 3 tool dm -3 HC1 dropwise to 67 gmol dm 3 sodium ABA. There was an isosbestic point at 258 rim. It was assumed that absorbance changes reflect protonation of the carboxyl group.
Results and Discussion The initial c h a r a c t e r i s a t i o n o f the b r o a d features o f A B A u p t a k e b y b e a n r o o t tissue was c a r r i e d o u t using p o o l e d segments. Subsequently, the r e s o l u t i o n o f the analysis was increased using segments cut f r o m particular r o o t zones.
Time Course and p H Dependence of [2-14C]ABA Uptake by Pooled Root Segments T h e u p t a k e o f [2-14C]ABA (1.0 g m o l d m - 3) by 4 m m long segments was f o l l o w e d (0-60 rain) at p H 4.0, 5.0 a n d 6.5 (Fig. 1). T h e u p t a k e is increased as the p H b e c o m e s m o r e acidic, a n d is initially r a p i d b u t then slows to a steady rate, a t t a i n e d earlier at h i g h e r p H values. T h e first p h a s e p r o b a b l y m a i n l y represents u p t a k e into cells at the cut surfaces in direct c o n t a c t with the i n c u b a t i o n m e d i u m . T h e p r o g r e s s i n g s t e a d y u p t a k e , which continues for at least a further h o u r at p H 5.0 will reflect c o n t i n u i n g p e n e t r a t i o n o f A B A to cells within the segment a n d also any m e t a b o l i c conversions. A B A is slowly m e t a b o l i s e d by r u n n e r b e a n r o o t tissue: in the ' W e i s s e r R i e s e ' variety used
1"0
f O'8
~
9
g0-~
2e~
0
0
I
I 20
I 30 Time / mi~
I
40
I
50
I 60
Fig. 1. Uptake of [2-1~C]ABA (1 Ilmol dm -3) into 4 mm pooled root segments (9 per incubation) from 3 d-old seedlings over 0 t o 6 0 m i n . - A - p H 4 . 0 ; - o - p H 5 . 0 ; - m pH6.5
by H a r t u n g a n d Behl (1974, 1975), m e t a b o l i s m o n l y b e c a m e significant after 11 h e x p o s u r e to r a d i o a c t i v e h o r m o n e , a l t h o u g h in our e x p e r i m e n t s a b o u t 2 0 % A B A was c o n v e r t e d to a less p o l a r substance after 2 h i n c u b a t i o n (Table 1). In s u b s e q u e n t experiments, i n c u b a t i o n times were generally limited to 5 min, to a p p r o x i m a t e to the initial rate o f u p t a k e , a n d when no d e t e c t a b l e m e t a b o l i s m o f A B A h a d o c c u r r e d (Table 1). A B A u p t a k e (5 min, 0.5 g m o l d m - 3) shows a sigm o i d a l d e p e n d e n c e on external p H (Fig. 2) over the range p H 3.5 to p H 7.0, with a m i d p o i n t at a b o u t p H 4.6, close to the p K o f A B A . Such b e h a v i o u r has p r e v i o u s l y been f o u n d for l i p o p h i l i c w e a k acids using c u l t u r e d cells (including A B A , R u b e r y u n p u b lished) a n d stem tissues ( R u b e r y a n d S h e l d r a k e i973; R u b e r y 1977; Davies a n d R u b e r y 1978) a n d is consistent with passive A B A H p e r m e a t i o n being a m a j o r c o m p o n e n t o f net u p t a k e . This i n t e r p r e t a t i o n is supp o r t e d b y the ability o f 2 , 4 - d i n i t r o p h e n o l a n d c a r b o n y l c y a n i d e p - t r i f l u o r o m e t h o x y p h e n y l h y d r a z o n e (Fig. 3) to decrease net A B A u p t a k e in a m a n n e r c o n s o n a n t with their a c t i o n as p r o t o n i o n o p h o r e s , t e n d i n g to equilibrate p H g r a d i e n t s across m e m b r a n e s . C o m p a r ison with rates of I A A u p t a k e b y r o o t segments (not shown) suggests t h a t I A A H is a b o u t 5 times m o r e p e r m e a n t t h a n A B A H . This is n o t u n e x p e c t e d since A B A is a larger a n d s o m e w h a t m o r e p o l a r m o l e c u l e
M.C. Astle and P.H. Rubery: ABA Uptake by Root Segments 1. Extent of metabolic conversions of [2-14C]ABA and[1-14C]benzoic acid by bean root segments. 4 mm segments were incubated at 25~ C in buffered (pH 5.0) uptake media; basal agar donor blocks were applied to 15 mm segments. Per-cent conversion to methanol-soluble radioactivity not identical with authentic markers after thin layer chromatography (see Materials and Methods) is shown Table
Root region incubated
Time
% 14C in metabolite
ABA
4 mm segments: sub-apical root hair region
Benzoic acid
5min 2h
undetectable" 23b, 25 ~
10 89
5min 2h
undetectable" 17d
91
I5 mm segments with basal donor blocks : sub-apical 12 h root hair region 12 h
47 ~ 51e
100 _
0.5 [tmol dm -3 [1-14C]benzoate was supplied. [2-a4C]ABA concentrations: a, 5 gmol dm-3 to accommodate short uptake time; b, 0.5 gmol dm 3; c, 0.5 gmol dm-3+5 gmol dm -3 nonradioactive ABA; d, 1.0 gmol dm 3; e, 2.5 gmol dm -3 to accommodate slower uptake from agar blocks
A
100
70
60
5O & ~o ~o
< 10 0
0
1
100
1000
[Additive]/uM
Fig. 3. The effects of 2,4-dinitrophenol (0 1.000 gmol d m - 3) ( o - ) and carbonylcyanide p-trifluoromethoxy phenylhydrazone (020 gmol dm 3) ( [] ) on [2-14CIABA uptake (0.5 gmol dm -3, 2 h,
pH 5.0) into 3 mm sub-apical segments (12 per incubation) from 3 d-old seedlings
with a lower ether/water p a r t i t i o n coefficient t h a n I A A ( C o l l a n d e r 1949; M i l b o r r o w 1967).
Concentration Dependence of ABA Uptake by Pooled Root Segments
90
A B A u p t a k e (0 160 g m o l d m -3) after 5 rain i n c u b a tion was d e t e r m i n e d at p H 4.0, 5.0 a n d 6.5. There was n o i n d i c a t i o n of overall s a t u r a t i o n at a n y of these p H values (Fig. 4). However, a saturable comp o n e n t does a p p e a r to c o n t r i b u t e significantly at the lower c o n c e n t r a t i o n s where A B A u p t a k e is relatively higher. This is also shown in the inset of Fig. 4 as the effect of increasing c o n c e n t r a t i o n s of n o n - r a d i o a c tive A B A (0.5-6,0 g m o l d m - 3 ) t o i n h i b i t u p t a k e of 0.5 g m o l d m 3 [2_Ir A (5 min) at p H 5.0 to a c o n s t a n t p l a t e a u value, representing s a t u r a t i o n of a putative carrier. The u p t a k e c o m p o n e n t insensitive to u n l a b e l l e d A B A reflects c o n t i n u i n g n o n - m e d i a t e d diffusion of A B A H , or possibly the o p e r a t i o n of a low affinity carrier system.
80
70
60
E 50 S0 30 20 10 0
315
I
I
I
I
I
3
4
5
6
7
pH
Fig. 2. pH-dependence of [2-1~C]ABA uptake (0.5 gmol dm 3,
Characteristics of ABA Uptake by Different Regions of the Root
5 min) expressed as per cent of maximum uptake (found by extrapolation to about pH 3). 10-4 mm segments from 4 d-old seedlings were used per incubation. Maximum uptakes were for pooled segments (-o-) 4.2 dpm mg- 1 5 rain 1; for sub-apical segments ( 9 -) 8.6 dpm mg -1 5 min-l; for root hair region segments (_A_) 3.7dpmmg-1 5min 1
In order to investigate the u n i f o r m i t y of A B A u p t a k e characteristics of the different regions of the root collected in the pooled segments, batches of 4 m m segm e n t s were assembled from specified regions (see ma-
316
M.C. Astle and P.H. Rubery: ABA Uptake by Root Segments Table 2. Effect of a ten-fold excess of nonradioactive ABA on uptake of [2-14C]ABA (5 rain, pH 5.0) by segments from different regions of primary roots of 4 d-old seedlings (0.5 gmol dm -3 labelled ABA) and lateral roots of 5 d-old seedlings (0.8 gmol dm 3 labelled ABA)
v=
~
1.6
Significance was assessed using Student's t test
30 ~
Root region incubated
0
7-
0
2
without [IZC]ABA
with [12C]ABA
Terminal 2 mm, including cap.
2.85__0.15 (3)
2.30_+0.11 (4)
Subapical 4 mm, i-2 mm tip removed.
3.74_+0.11 (3)
2.57+0.03 (3) (p < 0,001)
4 mm segments, immediately before root hair region.
1.85_+0.04 (4)
1.81_+0.09 (4) (ns)
4 mm segments from root hair region.
1.37_+0.03 (3)
1.35_+0.10 (4) (ns)
2 mm segments from stele of root hair region.
6.76_+0.13 (3)
6.44+_0.34 (4) (ns)
2 mm segments from cortex of root hair region.
2.83Z0.13 (4)
2.62_+0.tl (4) (ns)
Terminal 3 ram, including cap.
4.05_+0.tl (4)
3.32_+0.07 (4) (p
i
-5
[IZc- ABA]
/"pM
I
t
5
10
Fig. 5. Dixon plot showing dependence of the saturable component of [2-~4C]ABA uptake (0.8 ~tmol dm -3, pH 5.0. 5 rain) on nonradioactive ABA concentration. 12.3mm sub-apical segments from 3 d-old seedlings were used per incubation. Magnitude of [2A4C]ABA uptake due to saturable component was determined by subtraction of diffusive component (plateau level of [2-1SC]ABA uptake at saturating non-radioactive ABA concentrations) from measured [2A4C]ABA uptake. Line drawn by least squares linear regression, using a weighting factor of v 3
levels in maize root apices (up to 0.25 gmol kg -1 fresh wt - Rivier et al. 1977) and Phaseolus vulgaris roots (0.12 gmol kg -1 fresh wt - Weiler 1980). Further investigations of the mechanism and driving forces of carrier-mediated ABA transport are in progress; one possibility is cotransport, as suggested previously for the auxin uptake carrier present in crown gall cells (Rubery 1978). Attempts to Detect Polar Transport of ABA in Bean Root Tissue In view of the restriction of the ABA carrier to the apical regions, we investigated the occurrence of polar ABA transport in longer segments of the primary roots, both including and excluding the apical tissue. An initial study involved incubating subapical 14 mm segments (first 2 mm discarded) for 2 h in 0.5 gmol dm -3 [2-14C]ABA at pH 5.0, followed by measurement of radioactivity present in sequential 2 mm slices. The resulting profile showed a strong gradient of radioactivity towards the apical end, such as might be expected if acropetal transport were occurring. However, division of the tissue into 2 mm slices before incubation with ABA revealed a comparable gradient in uptake capacity (Fig. 6 a). When the same protocol was followed with the next 14 mm long portion of the root ("root hair region", 16-30 mm from the apex), the radioactivity was symmetrically distributed, with increased uptake adjacent to both cut surfaces.
318
M.C. Astle and P.H. Rubery: ABA Uptake by Root Segments
z~0
?-0
0'6
~1'0
