1494 The Plant Cell
LETTER TO THE EDITOR
The Role of Auxin in Plant Embryogenesis
In the June 1993 issue of THE PLANT
CELL, the research paper by Liu et al.
(1993) and the review article by Chasan
(1993) addressed the important question
of the role of auxin in plant embryogene-
sis. These articles are especially
noteworthybecausethey discuss the liter-
ature on both zygotic and somatic
embryos, each of which offers distinct ad-
vantages for studying the underlying
mechanismsof plant embryogenesis. The
intent of this letter isto place the work of
Liu et al. (1993) in the context of what is
already known about the transport, activ-
ity, and biosynthesis of auxin during plant
embryogenesis.
Polar auxin transport has long been
postulated to play a central role in plant
embryogenesis. Microscale auxin trans-
port assays have been used to show that
the hypocotyls of mature embryos dis-
sected from both gymnosperm and an-
giosperm seeds exhibit pronounced
polar auxin transport toward the root
end irrespective of their orientation
(Greenwood and Goldsmith, 1970; Fry and
Wangermann, 1976). This polar transport
is completely blocked by 10 VM 2,3,5-
triiodobenzoic acid (TlBA) (Greenwood
and Goldsmith, l m ) , which suggeststhat
the TIBA-mediated effects on cultured
zygotic embryos observed by Liu et al.
(1993) are directly attributable to the inhi-
bition of polar auxin transport asopposed
to being a secondary effect unrelated to
auxin transport. In addition, Fry and
Wangermann (1976) were the first to pro-
pose that the initiation of polarized auxin
transport in the globular embryo might
mediate the morphological polarity ex-
pressed in the later stages of plant
embryogenesis.
The possible role of polar auxin trans-
port in somatic embryogenesiswas tested
by Schiavone and Cooke (1987), who treat-
ed different stages of carrot somatic
embryos with TlBA and a different auxin
transport inhibitor, N-(l-naphthy1)phtha-
lamic acid (NPA). Both auxin transport transport inhibitors suggests a provoca-
inhibitors at a concentration of 1 pM are tive hypothesis about the role of auxin in
able to block the ability of somatic embryonic pattern formation. If we as-
embryos to undergo morphogenetictran- sume that these differences cannot be
sitions to the subsequent stages: globular ascribed to species differences or other
embryos undergo persistent spherical ex- trivial factors, then it seems reasonable
pansion, oblong embryos (an intermediate to hypothesize that embryonic pattern for-
stage in somatic embryogenesis)continue mation is actually regulated by two
axis elongation without any cotyledon ini- overlapping mechanisms: (1) a positional
tiation, and heart embryos develop mechanism that arises as a maternal ef-
additionalgrowth axes on their hypocotyls. fect from the ovular tissue surroundingthe
Further evidence that polar auxin trans- zygotic embryo or as a consequence of
port is regulating somatic embryo the polarized position of the egg cell
formation comes from microsurgical in- andlor early embryo within the embryo
vestigations, which have establishedthat sac, and (2) an auxin-mediated mecha-
cut pieces of somatic embryos can often nism that is established by the initiation
regenerateshoot structures from their ap- of polar auxin transport in the late globu-
ical ends and root structures from their lar embryo. Either mechanism is appar-
basal ends (Schiavone and Racusen, ently sufficient by itself to regulate the
1990, 1991). Because the regenera- initiation of the embryonic axis and the
tion process is sensitive to exogenous apical positioning of cotyledonstructures,
auxin as well as to transport inhibitors becausetransport inhibitos prevent these
(Schiavone, 1988), it is quite conceivable processes in somatic embryos, which
that the ability of developing somatic em- would seem to lack an active positional
bryos to maintainstructuralpolarity is also mechanism, but have no effect on zygot-
dependent on polar auxin transport. ic embryos, in which the positional
In contrast to Schiavone and Cooke's mechanism appears to persist even in em-
(1987) observations that carrot somatic bryo culture. Finally, the emergenceof two
embryos are profoundly affected by po- discrete cotyledons appears to be depen-
lar auxin transport inhibitors, Liu et al. dent solely on auxin transport because
(1993) reportedthat similarly treated zygot- the inhibitors block this process in both
ic embryos of Brassica juncea exhibit somatic and zygotic embryos. This
typical axis elongation and cotyledonposi- hypothesis of overlapping regulatory
tioning at the embryonic apex, but the mechanisms has important experimental
cotyledons emerge as a fused collarlike implications: the positional mechanism
structure around the apex rather than as can be manipulatedvia genetic or physi-
two discrete lateral structures. Their in- ological approaches in zygotic embryos
terpretation that a reduction in polar auxin only, whereas the auxin-mediated mech-
transport is responsiblefor the altered cot- anism may be investigated more easily in
yledon morphology was strengthened by somatic embryos, which lack the position-
their finding that the pinl-7 mutant of al mechanism, than in zygotic embryos.
Arabidopsis, which is known to have re- Liu et al. (1993) proposed two alterna-
duced auxin transport in its inflorescence tive models of localized auxin synthesis
(Okada et al., 1991), exhibits the same col- and subsequent transport to explain the
larlike cotyledon structure in its embryo. origin of the cotyledons at the end of the
We believe that the difference in the re- globular stage. Although the available evi-
sponses of carrot somatic embryos and dence on auxin biosynthesis during
6.juncea zygotic embryos to auxin polar somatic embryogenesis lacks the spatial

resolution neededto discriminate between
these two models, it does provide consid-
erable insight into how auxin gradients
may be established in developing em-
bryos. Using gas chromatography/mass
spectrometry, Michalczuk et al. (1992a)
demonstratedthat 2P-D-treated embryo-
genic callus (whose proembryogenic
masses may be considered the somatic
equivalent of preglobular embryos) has
extraordinary levels of endogenous aux-
in, i.e., approximately 60 ng/g f. wt. free
and 600 nglg f. wt. total IAA, which is syn-
thesized via a tryptophan-mediated
pathway (Michalczuk et al., 1992b). It is
difficult to imagine how effective auxingra-
dients could be establishedin preglobular
embryos in the presenceof such supraop-
timal concentrations.
Endogenous IAA levels in young somat-
ic embryos decline precipitouslyfollowing
transfer to 2,4-D-free medium, with the
result that postglobular embryos contain
between 15 to 30 nglg f. wt. of total IAA
(Michalczuk et al., 1992a), which is low
enough to establish interna1gradients of
auxin activity. More important, this IAA is
synthesized via a different, nontryptophan
pathway(Micha1czuketal., 1992b),which
implies that the induction of this second
pathway is required for the initiation of the
polar auxin transport thought to occur in
the apex of the late globular embryo
(Schiavone and Cooke, 1987; Liu et al.,
1993). Thus, isolating the genes for the
nontryptophan pathway may provide the
probes necessary to discriminate between
the two potential sites of auxin biosynthe-
sis predicted by the alternative models of
Liu et al. (1993).
Todd J. Cooke
Richard H. Racusen
Department of Botany
University of Maryland
College Park, MD 20742
Jerry D. Cohen
Horticultural Crops Quality Laboratory
USDA Beltsville Agricultura1
Research Center
Beltsville, MD 20705
REFERENCES
Chasan, R. (1993). Embryogenesis: New mo-
lecular insights. Plant Cell 5, 597-599.
Fry, S.C., and Wangermann, E. (1976). Polar
transport of auxin through embryos. New
Phytol. 77, 313-317.
Greenwood, M.S., and Goldsmith, M.H.M.
(1970). Polar transport and accumulation of
indole-3-aceticacid during root regeneration
by Pinus lambertiana embryos. Planta 95,
297-313.
Liu, C.-m., Xu, 2.-h., and Chua, N.-H. (1993).
Auxin polar transport is essential for the
November 1993 1495
LETTER TO THE EDITOR
establishment of bilateral symmetry during
early plant embryogenesis. Plant Cell 5,
621-630.
Michalczuk, L., Cooke, T.J., and Cohen, J.D.
(1992a). Auxin levels at different stages of
carrot embryogenesis. Phytochemistry 31,
1097-1103.
Michalczuk, L., Rlbnicky, D.M., Cooke, T.J.,
and Cohen, J.D. (1992b). Regulation of
indole-3aceticacid biosynthetic pathwaysin
carrot cell cultures. Plant Physiol. 100,
1346-1353.
Okada, K., Ueda, J., Komaki, M.K., Bell, C.J.,
and Shimura, Y. (1991). Requirement of the
auxin polar transport system in early stages
of Arabidopsis floral bud formation. Plant Cell
3, 677-684.
Schiavone, F.M. (1988). Micromanipulationof
somatic embryos of the domesticated carrot
reveals apical control of axis elongation
and root regeneration. Development 103,
657-664.
Schiavone, F.M., and Cooke, T.J. (1987). Un-
usual patterns of somatic embryogenesis in
domesticated carrot: Developmental effects
of exogenous auxins and auxin transport in-
hibitors. Cell. Diff. 21, 53-62.
Schiavone, F.M., and Racusen, R.H. (1990).
Microsurgery revealsregional capabilities for
pattern reestablishment in somatic carrot em-
bryos. Dev. Biol. 141, 211-219.
Schiavone, F.M., and Racusen, R.H. (1991).
Regeneration of the root pole in surgical-
ly transected carrot embryos occurs by
position-dependent,proximodistal replace-
ment of missing tissues. Development 113,
1305-1313.
 The Role of Auxin in Plant Embryogenesis
T. J. Cooke, R. H. Racusen and J. D. Cohen
PLANT CELL 1993;5;1494-1495
DOI: 10.1105/tpc.5.11.1494

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