| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
Plant Physiol. (1998) 116: 1505-1513
Dim-Red-Light-Induced Increase in Polar Auxin Transport in
Cucumber Seedlings1
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
ABSTRACT |
|---|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
We have developed and characterized a
system to analyze light effects on auxin transport independent of
photosynthetic effects. Polar transport of
[3H]indole-3-acetic acid through hypocotyl segments from
etiolated cucumber (Cucumis sativus L.) seedlings was
increased in seedlings grown in dim-red light (DRL) (0.5 µmol
m
2 s1) relative to seedlings grown in
darkness. Both transport velocity and transport intensity (export rate)
were increased by at least a factor of 2. Tissue formed in DRL
completely acquired the higher transport capacity within 50 h, but
tissue already differentiated in darkness acquired only a partial
increase in transport capacity within 50 h of DRL, indicating a
developmental window for light induction of commitment to changes in
auxin transport. This light-induced change probably manifests itself by
alteration of function of the auxin efflux carrier, as revealed using
specific transport inhibitors. Relative to dark controls, DRL-grown
seedlings were differentially less sensitive to two inhibitors of polar
auxin transport, N-(naphth-1-yl) phthalamic acid and
2,3,5-triiodobenzoic acid. On the basis of these data, we propose that
the auxin efflux carrier is a key target of light regulation during
photomorphogenesis.
Photomorphogenesis is the initiation of an ordered sequence of
events on time scales from seconds to weeks, regulating processes from
the level of protein and nucleic acid behavior to the differentiation of tissues and organs (Shropshire and Mohr, 1983 Polar transport of endogenous and applied auxins is a consistent
feature of plant stem and coleoptile tissue, and is closely tied to
development (Goldsmith, 1977 In grass seedlings both rapid effects of light on auxin transport
(Jones et al., 1991 One property that characterizes auxin-transport systems, and is
diagnostic of their function, is the susceptibility to specific inhibitors of polar auxin transport, all of which act at least in part
by blocking auxin efflux (Katekar and Geissler, 1980 From current understanding of the auxin efflux carrier, the differences
between rapid and long-term effects of light on polar auxin transport
can be explained in terms of two mechanisms: (a) fast, reversible
effects caused by light-induced changes in diffusible regulators of
efflux (such as flavonoids; see Jacobs and Rubery, 1988 However, one long-term consequence of photomorphogenesis, the
development of the photosynthetic apparatus, causes changes in polar
auxin transport related to energy status rather than a developmental
change in the auxin-transport system (Thimann and Wardlaw, 1963 We have characterized several important differences between auxin
transport in DG and DRL-grown cucumber (Cucumis sativus L.)
seedlings. DRL-grown seedlings showed approximately 2-fold increases in
transport velocities and intensities relative to DG controls.
Transferring seedlings between light environments indicated that the
acquisition and loss of the increased transport capabilities occurred
through slow developmental processes. The increase in transport was
accompanied by a decrease in sensitivity to polar auxin-transport
inhibitors, and the effect of light environment on responsiveness to
NPA and TIBA was not the same. These findings support the model
presented above for long-term changes in auxin transport caused by
modification of the auxin efflux carrier.
Plant Material and Light Treatments
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
). In plant stems photomorphogenesis has been described principally in terms of light-induced changes in elongation rate, and the biochemistry underlying these changes. The majority of progress has occurred in the
understanding of processes on shorter time scales and limited structural complexity. One mediator of light regulation of stem elongation is the plant hormone auxin (Behringer and Davies, 1992, and
refs. therein). A role for polar auxin transport in photomorphogenetic processes can be inferred from the effects of mutations affecting auxin
transport, including a variety of defects in photosynthetic, transport,
and growth capacities of mature plants (Carland and McHale, 1996;
Ruegger et al., 1997). These features of the form and function of
mature plants are the central outcomes of development directed by
photomorphogenesis.
; Thimann, 1977; Jacobs, 1979; Lomax et
al., 1995). Light-induced changes in transport correlate well with
rapid changes in stem elongation (Furuya et al., 1969; Jones et al.,
1991), and more gradual developmental changes, including vascular
differentiation, have been shown to be regulated by auxin transport
(Jacobs, 1979; Ruegger et al., 1997; see also introduction in Carland
and McHale, 1996). However, auxin transport itself has been described
as developmentally regulated (Jacobs, 1979). One example is the decline
of auxin transport with increasing tissue maturity in petiole and stem
tissue (Jacobs, 1979; Suttle, 1991).
) and long-term effects of light (Naqvi, 1975) have
been reported. When irradiations were given a short interval before the
auxin-transport assay, light caused a decrease in the amount of auxin
transported through tissue per unit time (transport intensity or export
rate), whereas the rate of movement of auxin (transport velocity) was
unchanged (Jones et al., 1991). Coleoptiles of corn seedlings grown for
long periods in WL exhibit transport velocities that are 50 to 60% of
those found in DG seedlings (Naqvi, 1975). There have been few studies
on the effects of light on auxin transport in dicot stems, and most of
these were confined to effects of light on LG seedlings. An early study
indicated that light had no effect on transport in DG pea (Thimann and
Wardlaw, 1963). Eliezer and Morris (1980) reported that WL illumination during the transport period caused a 35% increase in the velocity of
auxin transport in both DG and LG pea seedings, and that LG plants also
showed a 2-fold increase in transport intensity, but only when
transport occurred in the light.
; Lomax et al.,
1995; Delbarre et al., 1996). Developmental changes in responsiveness
to these inhibitors have been observed to occur in parallel with
changes in polar auxin transport (Suttle, 1991), and altered
developmental responses to these inhibitors have been used as screens
for mutations affecting auxin transport itself (Ruegger et al., 1997).
Chemical and physiological data suggest that synthetic inhibitors of
polar auxin transport: (a) have natural analogs that are flavonoid
molecules (Jacobs and Rubery, 1988), (b) may have several modes of
action (Katekar and Geissler, 1980; Bruun et al., 1992; Delbarre et
al., 1996), and (c) act on an efflux carrier target that may consist of
multiple components (Morris et al., 1991; Cox and Muday, 1994).
); and (b)
slower, less labile modification of the number, position, or function
of one or more components of the efflux carrier itself (Morris et al.,
1991). The finding that a mutation in a putative phosphatase regulatory
subunit gene alters auxin transport (Garbers et al., 1996) demonstrates
one possible pathway for efflux carrier regulation. In principle,
long-term responses can also be affected by diffusible regulators, and
modification of the efflux carrier could come about as a direct, if
delayed, regulatory effect of light or as a secondary consequence of
earlier light-regulated events influencing cell differentiation.
).
Distinguishing between the effects of the input of photosynthetic
energy on, and developmental regulation of, the auxin-transport system
is inherently difficult. To resolve this difficulty, we studied auxin
transport using an intermediate state of photomorphogenesis induced by
growth in continuous DRL (0.5 µmol m2
s1). Under DRL plants exhibit an altered
pattern of stem elongation, but an "etiolated" morphology is
preserved (Shinkle et al., 1992). Seedlings grown under DRL have closed
hooks, high hypocotyl elongation rates, and cotyledons that expand and
green minimally. Elongation of the hypocotyl differs from that of DG
seedlings in that the apical-to-basal graded decline in the elongation
rate seen in DG seedlings is not as steep in DRL-grown seedlings.
Hence, DRL-grown seedlings exhibit a photomorphogenetic change in
growth pattern, but do not develop photosynthetic competence, making it
possible to detect developmental changes in auxin transport.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
2 s1, as described by
Shinkle et al. (1992), or in similar growth rooms at the University of
North Carolina-Chapel Hill. Plants were grown for varying periods and
transferred between darkness and DRL as indicated below and in
``Results''.
Growth Measurements
Relative growth rates of specific regions of hypocotyls were determined as described in Shinkle et al. (1992). Regions of hypocotyls
were marked at 5-mm intervals with wet charcoal, and relative growth
rates (measured over a 2-h interval) were determined for marked
regions. Growth rates for the 10-mm regions used for transport analysis
described below were determined from combination and averaging relative
growth rates from the appropriate pair of 5-mm-marked regions.
Auxin-Transport Assays
Unless otherwise noted, chemicals were obtained from Sigma. NPA was obtained from Chemical Services (West Chester, PA).Method 1: Continuous Label Application and Collection Using Agar Blocks (Jacobs and Hertel, 1978)
Cylindrical agar-receiving blocks were prepared with 1.5% (w/v) 5 mm KH2PO4, pH 5.5, drawn into 3-mm i.d. glass tubes, and cut to 3-mm lengths. Donor blocks were made by adding 107 m
5-[3H]IAA (0.95 TBq
mmol1, Amersham) to partially cooled, but still
liquid, agar, which was then mixed by vortexing for 1 min. Donor and
receiver tubes were kept at 4°C until use. Transport assays were
performed on 5-mm segments from three regions of seedlings with
hypocotyls 5 to 6 cm long (taken at 94-98 h after planting for DG
seedlings and 106-110 h after planting for DRL-grown seedlings).
Apical segments were cut from within 0 to 10 mm from the hook, middle segments were cut from within the region 20 to 30 mm from the hook, and
basal segments were cut from within the region 40 to 50 mm from the
hook.
Method 2: Pulsed Application of Labeled IAA (Modified from Hasenstein [1987] and Jones et al. [1991])
Straight hypocotyls (5 cm) harvested from between seed attachment point and hook were cut and immediately placed apical end down in a 10-dram scintillation vial containing 250 µL of 3.8 × 107 m
5-[3H]IAA (2.5 µCi or 91.6 kBq) in 5 mm KH2PO4 and
30 mm Suc, pH 5.5 (transport buffer), and the vial was
capped to maintain high humidity. For time-course studies, after 15 min
of incubation, hypocotyls were removed from the vial, rinsed with
distilled water, cut to the apical 1 cm, and inserted vertically,
apical end down, in a 1% (w/v) agar slab (5 mm
KH2PO4 and 30 mm Suc, pH 5.5) kept in a humid chamber. Receiver blocks
made of the same agar were placed on the basal cut surface of the
hypocotyls and replaced at 30-min intervals. Blocks from three
hypocotyls for each time point and 1-cm hypocotyl segments were each
extracted in liquid-scintillation solution and counted as described.
For distribution of label at fixed times, the 5-cm hypocotyl segments
were handled identically, except that they were left intact, and after
the indicated time intervals were sectioned by cutting with a razor
blade into the following 5 regions, which were counted as described: 0 to 3 mm, 3 to 6 mm, 6 to 10 mm, 10 to 20 mm, and 20 mm and below.
Although transport in inverted tissue as used here has been shown to
differ from transport in upright tissue (see Jacobs, 1979), this does not affect the validity of the assay for comparisons between tissue types.
Method 3: Continuous Application of Label and Collection in Distal Tissue
Hypocotyls were handled as in method 2, except after 1 h, the apical 1 cm and basal 4 cm were separated and measured by liquid-scintillation spectroscopy as described. When tissue was treated with NPA or TIBA, these substances were added to the transport buffer and were present throughout the transport period. Transport was routinely expressed as the percentage of total cpm in a hypocotyl found in the basal segment. To test the effect of transfer of seedlings between darkness and DRL (Fig. 4), seedlings were marked with charcoal at the hook/hypocotyl boundary at the time of transfer. At 30 or 50 h after transfer, transport assays were done with segments cut from the most apical region of the hypocotyl and with segments cut from below the charcoal mark using the procedure described above. Controls for these transfers were seedlings grown in darkness and in DRL, marked at the same time as transferred seedlings and left in their original light regimes for the same period of time as transferred seedlings, before being used in transport experiments as above.
|
Identification of 3H-Labeled Compounds Transported through Tissue
Agar blocks containing transported radioactivity (accumulated over 1 h) from 12 segments were extracted in 500 µL of 100% ethanol for 1 h at room temperature. The extract was concentrated 5-fold in a Speed-Vac (Savant, Farmingdale, NY), then subjected to TLC using Kodak silica gel chromatograms and a solvent system consisting of ethylacetate:isopropanol:ammonium hydroxide (45:35:10, v/v). Chromatograms were cut into 24 sections and counted by liquid scintillation. As a control, 1 µL of 5-[3H]IAA from the original ethanol stock was placed in 25 µL of ethanol for 1 h at room temperature and chromatographed as above. Figure 1 shows that the [3H]IAA transported through the tissue into blocks was not catabolized. The peak in the high fraction numbers (22-23) is an oxidation product formed during TLC (Melhado et al., 1982). The chromatography data shown are for DG hypocotyl segments, but
similar results were obtained for DRL-grown tissue (data not shown).
Since hypocotyls were allowed to take up
[3H]IAA for 1 h, the radioactivity
collected is analogous to the radioactivity collected at the first four
points in the time course, and the conditions used for all subsequent
experiments. This indicates that whatever the final fate of the
[3H] label, the counts that crossed the
boundary between the apical 1 cm and either an agar block or basal
tissue most likely did so as [3H]IAA.
|
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Methods
Results
Discussion
References
|
|---|
Figure 2A shows the two different
growth distributions seen in DG and DRL-grown cucumber seedlings. The
decline in growth rate from apical to basal regions was greater in DG
seedlings, but as noted previously (Shinkle et al., 1992), the
elongation rate of the hypocotyl as a whole was very similar between
the two light regimes. Polar transport of
[3H]IAA for 3 h through 5-mm segments of
hypocotyls taken from the regions identified in Figure 2A also showed
an apical-to-basal decline in both types of tissue (Fig. 2B). The
results shown extend the well-established finding that stem elongation
rate and auxin-transport capacity are not always correlated (see
Jacobs, 1979). In both light conditions there was a decline in
transport rate along the apical to basal axis of the hypocotyl, as
observed previously (Suttle, 1991), but this pattern is clearly not
well correlated with the decline in growth rate along the apical-basal
axis.
|
The principal finding from these experiments is that
photomorphogenesis evokes a major change in the auxin-transport system in dicots, most likely by altering regulation of auxin efflux. DRL
causes cucumber seedlings to develop a 2-fold increase in transport
velocity, a 2-fold increase in transport intensity, and altered
responses to inhibitors of transport, relative to DG controls. Although
Table II shows that hypocotyls from DG seedings immobilize more counts
from [3H]IAA than do hypocotyls from DRL-grown
seedlings, the data do not distinguish between the possibility that
increased metabolism retards transport and the possibility that slower
transport allows greater opportunities for the action of pathways
metabolizing IAA. The latter explanation is more consistent with the
decrease in transport velocity and the change in sensitivity to
inhibitors, although these data in no way rule out a
contribution of altered auxin metabolism to the overall phenomenon.
Received August 21, 1997;
accepted December 22, 1997.
Abbreviations:
DG, dark-grown.
DRL, dim-red light.
LG, light-grown.
NPA, N-(naphth-1-yl)phthalamic acid.
TIBA, 2,3,5-tri-iodobenzoic acid.
WL, white light.
The technical assistance of Ms. Christiana Rodgers with
preliminary experiments is gratefully acknowledged. The majority of the
experiments were carried out in the laboratory of Dr. Alan Jones,
University of North Carolina at Chapel Hill, while J.R.S. was on
sabbatical.
Behringer FJ,
Davies PJ
(1992)
Indole-3-acetic acid levels after phytochrome-mediated changes in stem elongation rate of dark- and light-grown Pisum seedlings.
Planta
188:
85-92
[CrossRef]
Beyer EM,
Morgan PW
(1969)
Ethylene modification of an auxin pulse in cotton stem sections.
Plant Physiol
44:
1690-1694
Bruun SA,
Muday GK,
Haworth P
(1992)
Auxin transport and the interaction of phytotropins.
Plant Physiol
98:
101-107
Burg SP
(1973)
Ethylene and plant growth.
Proc Natl Acad Sci USA
70:
591-597
Carland FM,
McHale NA
(1996)
LOP1: a gene involved in auxin transport and vascular patterning in Arabidopsis.
Development
122:
1811-1819
[Abstract]
Cosgrove DJ
(1981)
Rapid suppression of growth by blue light.
Plant Physiol
67:
584-590
Cox DN,
Muday GK
(1994)
NPA binding activity is peripheral to the plasma membrane and is associated with the cytoskeleton.
Plant Cell
6:
1941-1953
Daniel SG,
Rayle DL,
Cleland RE
(1989)
Auxin physiology of the tomato mutant diageotropica.
Plant Physiol
91:
804-807
Delbarre A,
Muller P,
Imhoff V,
Guern J
(1996)
Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid, napthalene-1-acetic acid, and indole-3-acetic acid in suspension-cultured tobacco cells.
Planta
198:
532-541
[ISI]
Depta H,
Rubery PH
(1984)
A comparative study of carrier participation in the transport of 2,3,5-triiodobenzoic acid, and 2,4-dichlorophenoxyacetic acid by Cucurbita pepo L. hypocotyl segments.
J Plant Physiol
115:
371-387
[ISI]
Eleizer J,
Morris DA
(1980)
Cell length, light and 14C-labelled indol-3yl-acetic acid transport in Pisum sativum L. and Phaseolus vulgaris L.
Planta
149:
327-331
Furuya M,
Pjon CJ,
Fujii T,
Ito M
(1969)
Phytochrome action in Oryza sativa L. III. The separation of photoreceptive site and growing zone in coleoptiles, and auxin transport as effector system.
Dev Growth Differ
11:
62-76
[Medline]
Garbers C,
DeLong A,
Deruere J,
Bernasconi P,
Soll D
(1996)
A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.
EMBO J
15:
2115-2124
[ISI][Medline]
Goldsmith MHM
(1967)
Movement of pulses of labeled auxin in corn coleoptiles.
Plant Physiol
42:
258-263
Goldsmith MHM
(1977)
The polar transport of auxin.
Annu Rev Plant Physiol
28:
439-478
Hasenstein K-H
(1987)
Non-transportable radioactivity in hypocotyls of Helianthus annuus after application of [3H]-IAA: analysis of diffusion parameters.
Physiol Plant
70:
139-145
Jacobs M,
Hertel R
(1978)
Auxin binding to subcellular fractions from Cucurbita hypocotyls: in vitro evidence for an auxin transport carrier.
Planta
142:
1-10
Jacobs M,
Rubery PH
(1988)
Naturally occurring auxin transport regulators.
Science
241:
346-349
Jacobs WP (1979) Movements of hormones. In Plant
Hormones and Plant Development. Cambridge University Press, Cambridge,
UK, pp 195-243
Jones AM,
Cochran DS,
Lamerson MA,
Evans ML,
Cohen JD
(1991)
Red light-regulated growth.
Plant Physiol
97:
352-358
Katekar GF,
Geissler AE
(1980)
Auxin transport inhibitors. IV. Evidence of a common mode of action for a proposed class of auxin transport inhibitors: the phytotropins.
Plant Physiol
66:
1190-1195
Lomax TL,
Muday GK,
Rubery PH
(1995)
Auxin transport.
In
PJ Davies,
eds, Plant Hormones: Physiology, Biochemistry and Molecular Biology.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 509-530
Melhado LL,
Pearce CJ,
D'Alarco M,
Leonard NJ
(1982)
Specifically deuterated and tritiated auxins.
Phytochemistry
21:
2879-2885
[CrossRef]
Morris DA,
Rubery PH,
Jarman J,
Sabater M
(1991)
Effects of inhibitors of protein synthesis on transmembrane auxin transport in Cucurbita pepo L. hypocotyl segments.
J Exp Bot
42:
773-783
Naqvi SM
(1975)
Kinetics of auxin transport in light and in dark grown Zea mays L. coleoptile segments.
Z Pflanzenphysiol
76:
379-383
Rayle DL,
Ouitrakul R,
Hertel R
(1969)
Effects of auxins on the auxin transport system in coleoptiles.
Planta
87:
49-53
[CrossRef][ISI]
Rohwer F,
Schierle J
(1982)
Effect of light on ethylene production: red light enhancement of 1-aminocyclopropane-carboxylic acid concentration in etiolated pea shoots.
Z Pflanzenphysiol
107:
295-300
Ruegger M,
Dewey E,
Hobbie L,
Brown D,
Bernasconi P,
Turner J,
Muday G,
Estelle M
(1997)
Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects.
Plant Cell
9:
745-757
[Abstract]
Shinkle JR,
Jones RL
(1988)
Inhibition of stem elongation in Cucumis seedlings by blue light requires calcium.
Plant Physiol
86:
960-966
Shinkle JR,
Lomax-Reichert T,
Hertel R,
Briggs WR
(1982)
Correlation of in vivo and in vitro auxin transport in developing Cucurbita pepo hypocotyls.
Carnegie Inst Wash Year Book
81:
21-23
Shinkle JR,
Sooudi SK,
Jones RL
(1992)
Adaptation to dim-red-light leads to a non-gradient pattern of stem elongation in Cucumis seedlings.
Plant Physiol
99:
808-811
Shropshire W Jr,
Mohr H
(1983)
Photomorphogenesis. Encyclopedia of Plant Physiology, New Series, Vol 16A & 16B.
Springer Verlag, Berlin
Smith CW,
Jacobs WP
(1969)
The movement of IAA-C14 in the hypocotyl of Phaselous vulgaris.
Am J Bot
56:
492-497
Suttle JC
(1988)
Effects of ethylene treatment on polar IAA transport, net IAA uptake and specific binding of N-1-naphthylphthalamic acid in tissues and microsomes isolated from etiolated pea epicotyls.
Plant Physiol
88:
795-799
Suttle JC
(1991)
Biochemical bases for the loss of basipetal IAA transport with advancing physiological age in etiolated Helianthus hypocotyls.
Plant Physiol
96:
875-880
Thimann KV (1977) Polarity and transport. In Hormone
Action in the Whole Life of Plants. University of
Massachusetts Press, Amherst, MA, pp 71-92
Thimann KV,
Wardlaw IF
(1963)
The effect of light on the uptake and transport of indoleacetic acid in the green stem of the pea.
Physiol Plant
16:
368-377
View this table:
Table I.
Comparison of basipetal and acropetal transport of
[3H]IAA from agar blocks through hypocotyl sections from
DG and DRL-grown cucumber seedlings
Donor agar blocks containing 10 7 m
[3H]IAA were prepared in 5 mm
KH2PO4 buffer, pH 5.5, and placed on 5-mm
hypocotyl sections from the cut within the region from 20 to 30 mm from
the hook. Transport took place for 3 h as described in
``Materials and Methods''. Results show total cpm (donor plus
sections plus receivers), cpm in receivers, and cpm in sections, as
well as percent uptake (cpm in receivers plus sections) transported to
receivers. Results are the average of 6 to 14 replicate sections, and
are shown ± se.
1,
respectively), and the rationale for the design of subsequent experiments.
View larger version (14K):
[in a new window]
Figure 3.
Time course for movement of a pulse of
[3H]IAA through 1-cm segments of cucumber seedlings grown
in darkness or in DRL. Time 0 indicates the end of a 15-min immersion
of the apical end of 5-cm hypocotyl segments in a thin film of 30 mm Suc buffered to pH 5.5 with 5 mm
KH2PO4 containing 2.5 µCi of
[3H]IAA. Segments were then cut to 1 cm and receiver agar
blocks were placed on the basal ends. At 30-min intervals, blocks were removed for counting and replaced with fresh blocks. 
, DRL-grown seedlings; 
, DG seedlings, with experiments performed with minimal exposure to dim-green work light; and 
, DG seedlings with
experiments performed under room light. Data shown are means for 8 replicates for both of the experiments with DG seedlings, and 10 replicates for experiments with DRL-grown seedlings. Error bars
represent se.
View this table:
Table II.
Distribution 3H label along hypocotyl
axes of DG and DRL-grown cucumber seedlings given a 15-min pulse of
[3H]IAA
Hypocotyl sections of 5 cm were placed, apical end down, in 200 µL of
1% Suc buffered to pH 5.5 in 5 mm
KH2PO4 containing 3.8 × 10 7
m [3H]IAA (26 Ci/mmol) for 15 min, rinsed
with distilled water, and inserted, apical end down, in 1% distilled
agar containing 1% Suc buffered to pH 5.5 in 5 mm
KH2PO4. At 180 or 90 min, for DG and DRL-grown
tissue, respectively, sections were removed from agar and cut into
regions. Results shown are the average cpm of 28 and 16 replicate
sections for DG and DRL-grown tissue, respectively, and are shown ± se.
; Shinkle and Jones, 1988). The unlabeled
IAA caused a decline in the uptake of [3H]IAA
in both DG and DRL-grown tissue. Adding excess unlabeled IAA improves
transport efficiency in DG tissue, as reported previously (Rayle et
al., 1969), but the effect here was a modest one.
View this table:
Table III.
Effect of IAA concentration on basipetal transport
of [3H]IAA from buffered aqueous solution through the
first centimeter of 5-cm hypocotyl sections from DG and DRL-grown
cucumber seedlings
Sections were placed apical end down in 200 µL of 1% Suc buffered to
pH 5.5 in 5 mm KH2PO4 containing
3.8 × 10 7 m [3H]IAA (26 Ci/mmol) minus or plus 3.4 × 106 m cold
IAA. Transport took place for 1 h. Results shown are cpm in the
apical centimeter, cpm in the basal 4 cm, and percent of total cpm
taken up transported into the basal 4 cm. Results are the average of 12 to 25 replicate sections, and are shown ± se.
). At this time, seedlings showed
transport capacities intermediate between DG and DRL-grown controls in
both the apical and displaced regions. At 50 h after transfer, the
apical regions showed the same transport capacity as DRL-grown
controls, whereas the displaced region still showed a transport
capacity intermediate between DG and DRL-grown controls.
; Shinkle et al., 1982).
Seedlings grown in DRL lost their increased transport capacity slowly
as well, and complete loss only occurred in apical tissue (data not
shown).
) or of
the effect of the inhibitors on uptake (Delbarre et al., 1996). Hence,
saturation may not have been reached for the effects of inhibitors on
DRL-grown tissue.
View larger version (29K):
[in a new window]
Figure 5.
Concentration dependence of the effect of TIBA and
NPA on the transport of [3H]IAA through hypocotyls from
DG (A) and DRL-grown (B) cucumber seedlings. Apical ends of hypocotyl
segments were placed in a thin film of 30 mm Suc buffered
to pH 5.5 with 5 mm KH2PO4
containing 2.5 µCi of [3H]IAA and the indicated
concentrations of NPA or TIBA for 2 h. Transport is expressed as
percent of total counts taken up found in tissue basal to the first
centimeter (with calculation as shown in Table III). Hatched bars
represent the effect of NPA; black bars indicate the effect of TIBA.
Data shown are means of 13 to 39 replicate sections. Error bars
represent se. In all cases where error bars do not overlap,
Student's t tests show samples to be significantly
different at P < 0.05 or better.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
),
indicating that the change in auxin transport is unlikely to cause the
change in the growth pattern. Since the pattern of transport capacity
is not well correlated with the pattern of growth (Fig. 2), it seems
likely that light regulation of these two processes occurs by two
independent mechanisms.
; Lomax et al., 1995), and this polarity is preserved
in DRL-grown seedlings (Table I), the efflux carrier is of necessity a
target for up-regulation or derepression by DRL. Based on the changes
in response to inhibitors of auxin transport, the primary effect of
which is on the function of the efflux carrier (Lomax et al., 1995;
Delbarre et al., 1996), the efflux carrier may be a principal target of
the light-induced change in transport.
. We believe our results are the first
demonstration of an in vivo separation of responses to NPA and TIBA,
and thus our results are an important extension of the in vitro
evidence that NPA and TIBA do not compete for the same binding site
(Bruun et al., 1992). Since the uptake of TIBA may be carrier mediated
(Depta and Rubery, 1984), it is conceivable that light differentially
affects the uptake of these two inhibitors. It is more likely, since
there appear to be several proteins involved in the function of the
efflux carrier (Morris et al., 1991; Cox and Muday, 1994), that the
effect of DRL could alter one or more of these components in a manner
that differentially affects the response of efflux carrier to the two
inhibitors.
1. This is consistent with
our determination of transport velocity in DG plants from the time at
which labeled IAA is first detectable. The relatively low rate of total
transport of labeled IAA has been observed previously in DG bean (Smith
and Jacobs, 1969) and zucchini (Shinkle et al., 1982). The movement of
a coherent pulse of [3H]IAA shown for DRL-grown
cucumber hypocotyls has been reported in both DG corn coleoptiles
(Goldsmith, 1967) and mesocotyls (Jones et al., 1991) This type of
result has also been reported in at least two species of LG dicots
(Beyer and Morgan, 1969; Daniel et al., 1989).
; Suttle, 1988). In addition, although
we grew seedlings in boxes with minimal gas exchange, we did not
observe the characteristic effects of ethylene on growth. Further, we
grew some batches of DG cucumber seedlings in open containers, and
others in our standard containers alongside pea seedlings, plants known
to produce physiologically relevant amounts of ethylene (Rohwer and
Schierle, 1982). The open-container-grown seedlings did not show any
increase in transport, whereas the seedlings grown under presumably
increased ethylene levels showed both morphological effects and
inhibition of auxin transport (data not shown). The long time required
for DG seedlings to acquire increased transport capacity when
transferred to DRL (Fig. 4) also mitigates against ethylene as a
mediator of the effect.
; Jones et al., 1991), and long-term light
treatment was found to cause a decrease in transport velocity (Naqvi,
1975). Our results also differ from those found in the one study of
light effects on auxin transport in dicots that we found (Eliezer and
Morris, 1980). Light during the transport period caused an increase in
transport velocity in DG seedlings of pea and bean, but had no effect
on transport intensity. Plants grown for long periods in WL exhibited
less than a 10% increase in transport velocity, and a 2-fold increase
in transport intensity in pea, but only when transport experiments were
also performed in the light. In our study long-term growth in DRL
caused at least a 2-fold increase in both transport velocity and
transport intensity, and performing transport assays in the light had
no effect at all on DRL-grown tissue (Fig. 3). Preliminary experiments
indicate that pea and tomato show the same type of response to DRL
(data not shown), suggesting that the DRL-induced increase in transport is a consistent feature of dicot photomorphogenesis and that WL-grown plants represent a different developmental state.
1
This work was supported by the U.S. Department
of Agriculture-National Research Inititiative Competitive Grants
Program (grant no. 93-37311-9590), a Faculty Development grant from
Trinity University to J.R.S., and an undergraduate research fellowship
to R.K. from the Pew Charitable Trusts through the Pew Mid-States
Consortium for Science and Mathematics.
FOOTNOTES
*
Corresponding author; e-mail jshinkle{at}trinity.edu; fax
1-210-736-7229.
ABBREVIATIONS
ACKNOWLEDGMENTS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
Copyright Clearance Center: 0032-0889/98/116/1505/09
© 1998 American Society of Plant Physiologists
This article has been cited by other articles:
|
|
 |
|
  G. Choi, J.-I. Kim, S.-W. Hong, B. Shin, G. Choi, J. J. Blakeslee, A. S. Murphy, Y. W. Seo, K. Kim, E.-J. Koh, et al. A Possible Role for NDPK2 in the Regulation of Auxin-mediated Responses for Plant Growth and Development Plant Cell Physiol., August 1, 2005; 46(8): 1246 - 1254. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Lin and H. Wang Two Homologous ATP-Binding Cassette Transporter Proteins, AtMDR1 and AtPGP1, Regulate Arabidopsis Photomorphogenesis and Root Development by Mediating Polar Auxin Transport Plant Physiology, June 1, 2005; 138(2): 949 - 964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. P. d. Santos, E. Purgatto, H. Mercier, and M. S. Buckeridge The Control of Storage Xyloglucan Mobilization in Cotyledons of Hymenaea courbaril Plant Physiology, May 1, 2004; 135(1): 287 - 299. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Gil, E. Dewey, J. Friml, Y. Zhao, K. C. Snowden, J. Putterill, K. Palme, M. Estelle, and J. Chory BIG: a calossin-like protein required for polar auxin transport in Arabidopsis Genes & Dev., August 1, 2001; 15(15): 1985 - 1997. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Stowe-Evans, D. R. Luesse, and E. Liscum The Enhancement of Phototropin-Induced Phototropic Curvature in Arabidopsis Occurs via a Photoreversible Phytochrome A-Dependent Modulation of Auxin Responsiveness Plant Physiology, June 1, 2001; 126(2): 826 - 834. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Lobello, M. Fambrini, R. Baraldi, B. Lercari, and C. Pugliesi Hormonal influence on photocontrol of the protandry in the genus Helianthus J. Exp. Bot., August 1, 2000; 51(349): 1403 - 1412. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||
