The polycotyledon Mutant of Tomato Shows Enhanced

Polar Auxin Transport1

Arif S.A. Al-Hammadi2, Yellamaraju Sreelakshmi2, Sangeeta Negi, Imran Siddiqi, and
Rameshwar Sharma*
School of Life Sciences, University of Hyderabad, Hyderabad–500046, India (A.S.A.A.-H., Y.S., S.N., R.S.);
and Center for Cellular and Molecular Biology, Tarnaka, Uppal Road, Hyderabad–500007, India (I.S.)

The polycotyledon mutant of tomato (Lycopersicon esculentum L. cv Ailsa Craig) showed altered development during
embryogenesis and during vegetative and reproductive phases. The phenotype was pleiotropic and included the formation
of extra cotyledons, changes in leaf shape, increased number of flowers (indeterminacy) with abnormal floral organs, the
formation of epiphyllous structures, and altered gravitropism. The earliest defects were observed at the transition from the
globular to the heart stage of embryogenesis with the formation of multiple cotyledons. Epidermal cells in the mutant
embryo were smaller and less expanded compared with wild type. Examination of polar auxin transport (PAT) showed a
striking enhancement in the case of the mutant. Increase in PAT did not appear to be caused by a decrease in flavonoids
because the mutant had normal flavonoid levels. Application of 2,3,5-triiodobenzoic acid, an inhibitor of polar transport of
auxin, rescued postgermination phenotypes of young seedlings. Our analysis reveals a level of control that negatively
regulates PAT in tomato and its contribution to plant development and organogenesis.

In higher plants, phytohormone auxin (indole-3- cells (Jacobs and Gilbert, 1983). The selective efflux of
acetic acid [IAA]) is transported basipetally from its auxin anion from the basal ends of transporting cells
site of synthesis at the shoot apex toward the roots by and arrangement of these cells in a long file from the
a process termed polar auxin transport (PAT). PAT shoot apex to the root apex is the basis of PAT.
provides directional information regulating several Much of our current knowledge about the nature of
facets of plant development such as cell elongation, components participating in PAT comes from molec-
vascular differentiation, apical dominance, tropic ular genetic analysis of mutants of Arabidopsis that
movements, and organ development (Lomax et al., are defective in transport of auxin. The pin1 mutant
1995). Physiological studies have indicated that PAT has reduced PAT, characteristically develops a naked
requires specific auxin influx and efflux carriers lo- pin-like inflorescence, and shows morphological ab-
cated on the plasma membrane of transporting cells. normalities in flowers and leaves. The PIN1 gene
Biochemical studies support a “chemiosmotic encodes a membrane protein that most likely func-
model” of auxin transport that proposes that un- tions as an auxin efflux carrier as suggested by its
charged, protonated auxin can enter cells from the localization at the basal ends of xylem parenchyma
acidic apoplast either passively by diffusion or via cells in vascular bundles (Gälweiler et al., 1998). An-
other group of mutants defective in the pin2/agr1/eir1/
energized uptake by specific influx carriers. In the
wav6 locus displays agravitropic roots and reduced
cytosol because of the more basic pH, IAA is depro-
root growth and exhibits a defect in auxin transport
tonated and is trapped within the cell due to poor
in roots. The product of the EIR1/AGR1/PIN2/WAV6
membrane permeability of anion. As a consequence,
gene shows similarities to PIN1 protein and is asym-
anionic IAA can leave the cell only by the action of metrically localized at the periclinal side of epider-
auxin efflux carriers (Rubery and Sheldrake, 1974; mal and cortical cells in the meristematic region and
Raven, 1975). The polarity of auxin transport pre- elongation zone of the root (Chen et al., 1998; Lus-
sumably is maintained by localization of auxin efflux chnig et al., 1998; Müller et al., 1998). Similar to pin1,
carrier molecules at the basal ends of transporting the pid1 mutant also shows reduced PAT and pro-
duces a naked inflorescence devoid of floral buds
1
This work was supported by the government of Yemen (fel- (Bennett et al., 1995). PID1 encodes a Ser-Thr protein
lowship to A.S.A.A.-H.), by Council of Scientific and Industrial kinase that was initially proposed to have a signaling
Research (CSIR; fellowships to Y.S. and S.N. and grant to R.S.), by or regulatory function in auxin action (Christensen et
International Atomic Energy Agency (IAEA; grant to R.S.), and by
al., 2000) and appears to act as a positive regulator of
Department of Science and Technology (DST; grant to R.S.).
2
These authors contributed equally to the paper.
auxin transport (Benjamins et al., 2001). A similar
* Corresponding author; e-mail [email protected]; fax link between auxin transport and protein phospha-
0091– 40 –23010120. tase 2A is seen in the rcn1 mutant, which shows root
Article, publication date, and citation information can be found curling in the presence of 1-naphthylphthalamic acid
at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.025478. (NPA; Rashotte et al., 2001). The aux1 mutant is de-
Plant Physiology, September 2003, Vol. 133, pp. 113–125, www.plantphysiol.org © 2003 American Society of Plant Biologists 113
Al-Hammadi et al.

fective in auxin uptake and displays defects in grav- 1999). Apparently, GNOM localizes to endosomes
itropic responses and resistance to exogenous auxin where it controls the polarized trafficking of PIN1 to
(Pickett et al., 1990). The AUX1 gene encodes an the basal plasma membrane (Geldner et al., 2003). In
influx carrier of auxin that has characteristics of an the gnom mutant, PIN1 accumulates internally and
amino acid permease-like protein (Bennett et al., fails to localize to the plasma membrane, causing the
1996; Marchant et al., 1999). mutant phenotype.
The treatment of plants with inhibitors of auxin Little information is available on mutants defective
efflux carrier activity, 2,3,5-triiodobenzoic acid in auxin action/transport in species other than Ara-
(TIBA) and NPA, influences many growth and de- bidopsis. The dgt mutant of tomato (Lycopersicon es-
velopmental processes thought to be controlled by culentum L. cv Ailsa Craig) has been reported to be
PAT. The pin1 phenotype could be copied in wild- defective in auxin action, and its phenotype can be
type Arabidopsis by treatment with auxin transport ascribed to reduced sensitivity of mutant tissues to
inhibitors with alterations in vascular development auxin (Kelly and Bradford, 1986; Muday et al., 1995;
and the formation of a pin-like inflorescence instead Coenen et al., 2003). In this report, we show that the
of floral buds (Okada et al., 1991; Gälweiler et al., poc (polycotyledon) mutation in tomato enhances PAT.
1998). It has been proposed that NPA acts by binding The mutant displays several developmental abnor-
to a plasma membrane-associated protein called the malities throughout the life cycle of the plants, from
NPA-binding protein (Muday et al., 1993; Bernasconi embryonic to vegetative and reproductive phases,
et al., 1996). Evidence suggests that NPA-binding suggesting that these changes may be related to al-
protein is distinct from the auxin efflux carrier and teration of auxin transport in the mutant. We also
plays an important role in cycling of auxin efflux show that the postgermination seedling phenotype of
carriers to plasma membranes (Geldner et al., 2001). the mutant can be rescued by treatment with TIBA
The tir3 mutants that are resistant to the inhibitory consistent with the view that this is due to enhanced
action of NPA on root elongation show a pleiotropic PAT.
phenotype, including reduced inflorescence height
with few and short siliques, decreased petiole and RESULTS
root length, and reduced apical dominance (Ruegger
et al., 1997). The TIR3 gene, renamed BIG, encodes a Genetic Characterization of the Polycot Mutant
protein that might be essential for proper positioning
The polycot mutants were initially identified based
of PIN1 at the plasma membrane (Gil et al., 2001). on their abnormal seedling phenotype highlighted by
Several lines of physiological evidence have sug- the presence of extra cotyledons. The mutants were
gested that a specific class of flavonoids may act as male sterile due to the lack of dehiscence of the
auxin transport inhibitors (Jacobs and Rubery, 1988). anther sac; nevertheless the anther sacs contained
Flavonoids such as quercetin act as competitive in- viable pollens (75%–80% germination). As a conse-
hibitors of NPA binding, suggesting both com- quence, the mutant was maintained in homozygous
pounds may regulate auxin transport by binding to state by manual self-pollination. The crosses with
the same protein (Murphy et al., 2000). The wild type yielded normal F1 seedlings with the wild-
flavonoid-deficient tt4 mutant shows reduced inflo- type phenotype in seedling stage and during vegeta-
rescence length, reduced apical dominance, increased tive and reproductive development and were self-
root branching, and stimulation of basipetal trans- pollinating with normal setting of seeds. The genetic
port of auxin in inflorescence segments (Brown et al., segregation analysis of F2 seedlings showed that the
2001). mutant phenotype was inherited as a monogenic
A distinct role for PAT has been noticed during Mendelian trait (119 seedlings, wild type; 42 seed-
plant embryogenesis, particularly in cotyledon devel- lings, polycot). The reciprocal crosses with wild type
opment. It is observed that auxin transport is neces- also showed 3:1 segregation of wild type and poc
sary for the establishment of bilateral symmetry dur- phenotype in F2 generation. The above segregation
ing the transition from the globular to heart stage of analysis suggests that the poc mutant contains a mo-
embryogenesis leading to formation of cotyledons nogenic, recessive, and nuclear mutation. Though we
(Liu et al., 1993; Hadfi et al., 1998). The treatment of obtained nine mutant lines, the results of comple-
excised globular mustard (Brassica juncea) embryos mentation analysis using homozygous poc lines
with inhibitors of PAT caused fusion of cotyledons at showed that all these lines were allelic, showing only
the subsequent heart stage (Liu et al., 1993). During the poc phenotype in the F1 generation. This was
wild-type embryogenesis, PIN1 shows polar localiza- further supported by the results of crossing with
tion beginning at the mid-globular stage with local- heterozygous poc lines. The F1 progeny of these lines
ization narrowing down to vascular precursor cells in segregated in typical 3:1 ratio of wild-type and poc
the cotyledonary primordia and embryo axis, seedlings (data not shown). Given the ratio of M2:M1
whereas the gnom mutant that shows disorganized plants (50,000:1,500), these results are consistent with
localization of PIN1 in globular embryos fails to de- the view that the above nine lines of poc mutants
velop cotyledonary primordia (Steinmann et al., likely represent siblings. One line was named poc1-1
114 Plant Physiol. Vol. 133, 2003
 Polar Auxin Transport in Tomato

and backcrossed twice for further characterization. simple cyme pattern. This pattern of organization in
The crossing of poc mutants with the Ds transposon- the inflorescence was disrupted in poc mutants, with
tagged dem mutant (Keddie et al., 1998), which also appearance of multiple branches on the main axis of
has a variable number of cotyledons, revealed lack of the inflorescence, which either terminated in a flower
allellism between these two mutants (M. Kavitha, or dichotomously branched further to give additional
M.S. Sharada, P. Janila, S. Negi, R. Sharma, unpub- flowers. The number of flowers in poc inflorescences
lished data). ranged between 20 and 60, with about 50 flowers
being more common (Fig. 1G). Markedly, in the poc
inflorescence, most flowers bloomed at the same
The poc Mutant Shows Pleiotropic time.
Developmental Defects The poc mutation showed incomplete penetrance
for the observed floral abnormalities (Tables I and II);
Nearly all poc mutant seedlings (98.5%, P ⬎ 0.0001) however, the penetrance for male sterility was nearly
showed extra cotyledons with approximately one- 100%. The poc mutants grown in the greenhouse or
half of the seedlings being tetracot and about 35% net house did not produce any fruit unless these were
seedlings being tricot. In the remaining seedlings, the self-pollinated by hand. Several abnormalities were
cotyledons showed varied extent of fusion yielding observed in poc floral organs such as increase in
tricot or dicot seedlings (Fig. 1A). The fused cotyle- number, decrease in size, and fusion of organs (Fig.
dons could be distinguished from a normal single 1H). Many flowers also showed occasional formation
cotyledon by the presence of two midveins in the of mosaic organs (Table II). The number of petals in
blade pointing toward the respective tips and run- poc mutant was higher, and petals were relatively
ning in parallel in the petiole (Fig. 1B). Though a short with petals of class C being smallest in size. In
minor number (1.5%) of seedlings showed a dicot nearly one-half of the flowers, the petals developed
phenotype, these could be distinguished from the an appendage of tissue facing toward stamens (Fig.
wild type by their round-shaped cotyledons (Fig. 1I). In many flowers, this appendage penetrated be-
1C). The roots of poc seedlings were distinctly shorter tween anther filaments, causing separation of sta-
than the wild type and showed earlier appearance of mens. Nearly two-thirds of flowers of the class C
lateral roots, and in adult mutant plants, the roots mutant flowers contained a patch of green tissue
were bushier than the wild type. resembling sepals in the center of petals. Similarly,
Nearly all poc mutant plants showed abnormalities the sepals of poc flowers showed an increase in num-
in the shape and size of leaves. Based on the leaf ber and about 5% to 8% of flowers contained petaloid
abnormalities, we classified poc plants under three sepals with a sector of tissue resembling petals
classes (Fig. 1D). The class A plants showed a short (Fig. 1J). Occasionally, the sepals were fused with
and bushy phenotype. Leaves of class A were shorter neighbors, with highest fusion (94%) in the flowers
in size and bore smaller, narrow, and curled leaflets of class C.
(Fig. 1E). A distinct feature of this class is the appear- The characteristic fusion of stamens resulting in the
ance of epiphyllous structures with a nearly 100% formation of a narrow-necked anther tube was nearly
frequency resembling leaves and shoots on rachis absent in the poc flowers (Fig. 1K). The flowers of the
normally near the junction to petiolules (Fig. 1F). The poc mutant were male sterile because the anthers
class B plants were only somewhat shorter than the lacked dehiscence. In class B and C flowers, the sta-
wild type. Though the leaves of class B appeared mens were twisted, distorted, and also variable in
similar to class A, the leaf size was intermediate size. In class C, the stamen number was reduced, and
between wild type and class A, and the leaflets were stamens were fused to the carpels (Fig. 1L). Despite
less curled than class A (Fig. 1E). Also, no epiphyl- these severe abnormalities, the gynoecium of these
lous structures were seen on class B leaves. The most flowers remained functional. Occasionally, from
severe abnormalities were observed in leaves of class within a fully differentiated poc flower, either an
C. The leaf lacked the small and minor leaflets, and inflorescence bearing flowers (Fig. 1M) or a shoot
the characteristic lobing of the blade was absent in developed. Several of these phenotypes are indica-
the major leaflets (Fig. 1E). In extreme cases, the leaf tive of the floral meristem retaining features of the
lacked both small and minor leaflets and formed a inflorescence and may reflect an incomplete transi-
lanceolate leaf. The loss of lobing in the leaflet mar- tion from the inflorescence to a floral meristem.
gin was associated with an altered venation pattern
of the leaflets with veins headed toward the leaflet
tip. The formation of first inflorescence in class C is Multiple Cotyledons Initiate at the Heart Stage in
delayed, and it appears only after formation of the Mutant Embryos
16th node, whereas in wild type and in the other two
classes of poc mutant, inflorescence forms between The appearance of multiple cotyledons in poc seed-
the eighth and 10th node. lings is a manifestation of the alteration of embryo
In wild-type plants, the inflorescence consists of development in the mutant. Growth of the embryo in
about seven to 14 flowers that are organized in a the poc mutant was slower than in the wild type. At
Plant Physiol. Vol. 133, 2003 115
Al-Hammadi et al.

Figure 1. Morphology of poc plants throughout life cycle. A, poc seedlings (clockwise from top right): wild-type seedlings
(arrow); dicot [1⫹(2)], note two midveins; dicot with two halves of curled cotyledons; dicot [1⫹(2)] with a partially fused
cotyledon; tetracot; and tricot. B, Fused poc cotyledons showing two midveins running parallel in the petiole. Wild type
(left). C, poc dicot seedling showing smaller and more rounded cotyledons. Wild type (left). D, Phenotype of 3.5-month-old
plants. Wild type (left), poc A (second from left), poc B (third from left), poc C (right). E, Adult leaves (sixth node onwards)
of 3-month-old wild type and poc mutant. In poc mutant, the leaf abnormalities vary from the simple lanceolate leaf to leaf
with variable number of leaflets, reduction in size, and loss of lobing in leaflets. Wild type (upper and lower left), poc B
(upper row), poc A (middle row), poc C (lower row). Wild-type leaves (upper and lower left). F, Epiphyllous structures
(arrows) on poc leaves appearing near the junction of petiolule to the rachis. G, poc inflorescence (right) showing multiple
blooming flowers with abnormal phyllotactic arrangement. H, poc flower (right) showing the absence of anther cone and
shorter petals. I, poc petal (right) with an appendage on stamen-facing side (arrow). J, poc sepal (right) with a petal-like
sector. Note the absence of trichomes (arrow). K, Twisted and short stamens of poc mutant lacking fusion to form anther
cone. Wild type (left). L, Fusion of stamens of poc to the carpel. M, Appearance of a new inflorescence from inside of a fully
differentiated flower of poc mutant (arrow).

116 Plant Physiol. Vol. 133, 2003

 Polar Auxin Transport in Tomato

Table I. The no. and length of floral organs in wild type (WT) and poc mutant
The sample size was 50 flowers from each genotype WT, poc A, poc B, and poc C. The P values (WT versus mutant) were calculated using
a two-tailed Student’s t test as described in “Materials and Methods.” The P values for all mutant samples were less than 0.0001.
No. of Organs Length of Organs (mm)
Organ
WT poc A poc B poc C WT poc A poc B poc C

Sepals 6.32 ⫾ 0.12 7.42 ⫾ 0.1 7.26 ⫾ 0.14 7.44 ⫾ 0.15 6.84 ⫾ 0.18 5.1 ⫾ 0.12 6.06 ⫾ 0.15 5.86 ⫾ 0.22
Petals 5.98 ⫾ 0.099 7.14 ⫾ 0.097 6.85 ⫾ 0.14 6.92 ⫾ 0.14 11.32 ⫾ 0.21 9.72 ⫾ 0.195 10.82 ⫾ 0.22 8.48 ⫾ 0.22
Stamens 6.04 ⫾ 0.09 7.04 ⫾ 0.08 6.84 ⫾ 0.13 5.4 ⫾ 0.16 8.28 ⫾ 0.14 7.38 ⫾ 0.14 7.9 ⫾ 0.13 6.08 ⫾ 0.18
Carpels 2.12 ⫾ 0.15 2.5 ⫾ 0.08 2.62 ⫾ 0.12 Not observed 7.26 ⫾ 0.13 6.4 ⫾ 0.14 7.56 ⫾ 0.12 5.2 ⫾ 0.18

10 d after pollination (DAP), the globular embryo of mutant cells (2,050 ⫾ 74.3 ␮m2, P ⫽ 0.0380; Fig. 3B).
the mutant was approximately 60% the size of the A similar difference in width was also visible in
wild-type embryo (Fig. 2, A and B). The poc embryo epidermal hypocotyl cells of light-grown plants, be-
was smaller at the late globular stage (Fig. 2, C and tween wild type (23.94 ⫾ 0.7 ␮m) and poc (15.13 ⫾
D). The slower development of poc embryos was 0.61 ␮m, P ⫽ 0.0007; Fig. 3, C and D).
apparent at the triangular stage, where the poc em-
bryo was shorter and squatter in shape relative to
the wild type (Fig. 2, E and F). The central procam- PAT Is Enhanced in the poc Mutant
bium region of the poc embryo was also broader at The transport of [14C] IAA was monitored in stem
this stage (Fig. 2, E and F). A comparison of cell segments in both the acropetal and basipetal direc-
shape in the medial epidermis of the poc embryo tions using two different methods outlined by Okada
revealed that the cells were more square in shape in et al. (1991) and Daniel et al. (1989; Fig. 4, A and B).
comparison with wild type, in which cells were Using both of these methods, it was found that the
more rectangular and elongated along the anticlinal polar transport of auxin in poc mutant was signifi-
axis (Fig. 2, I and J). In fact, poc embryos do not cantly higher than the wild-type control. In the acro-
show a typical heart stage because in place of two petal direction, transport of auxin was minimal and
cotyledons, three or four cotyledons form in the about the same in wild type and mutant. In contrast,
embryo (Fig. 2, G and H). a significant increase in auxin flow was observed in
the basipetal direction with the poc mutant display-
Cell Size Is Altered in the poc Mutant ing nearly 2.5- to 3-fold higher transport than the
wild type. The inclusion of TIBA reduced the mag-
A decrease in size of cells in the poc mutant was nitude of transport in both mutant and wild type. In
noticed for developing embryos at the triangular an assay for retention of auxin, the poc mutant dis-
stage. Scanning electron microscopy (SEM) examina- played decreased accumulation of auxin as evident
tion of the surface of cotyledons and hypocotyls by less retention of [14C] IAA as compared with wild
showed a similar change in cell size in the mutant type (Fig. 4C). The fact that inclusion of TIBA in-
seedlings. The increase in cotyledon number was creased the amount of retention of [14C] IAA indi-
associated with a reduction in size of epidermal cells cates the process to be mediated by PAT. The possi-
of cotyledons and hypocotyls for poc. The abaxial bility that enhanced PAT could be due to a reduction
epidermal cells of wild-type cotyledons (Fig. 3A) in flavonoid levels is argued against by the observa-
were larger in size (2,396 ⫾ 107.3 ␮m2) than the poc tion that both poc mutant (0.70 ⫾ 0.11, A330/shoot,
P ⫽ 0.7071) and wild type (0.64 ⫾ 0.10, A330/shoot)
Table II. The frequency of floral organ abnormalities in the flowers plants have similar levels and spectra of flavonoids
of different classes of poc mutant (n ⫽ 150 –200) (Fig. 4D).
Mutant Class
Phenotypic Abnormalities
poc A poc B poc C The Seedling Phenotype of poc Can Be
% Rescued by TIBA
Organ fusion
Fusion of sepals 5 5 94
The hypocotyls of dark-grown poc seedlings were
Lack of fusion of stamens 85 75 100 distinctly shorter than those of wild type (Fig. 5A). In
Stamens fused to carpels 0 0 89.4 view of enhanced PAT in the mutant, we reasoned
Petals and stamens fused 0 0 3 that faster PAT might deplete auxin levels in hypo-
to carpels cotyl cells, leading to the observed phenotype. In that
Identity change case, a reduction in PAT by application of TIBA
Petaloid sepals 7.5 7.5 5.3 should rescue the phenotype of poc seedlings. The
Outgrowth in petals 52.5 22.5 60 observed increase in length of hypocotyls of dark-
Sepaloid petals 0 2.5 65.8 grown poc seedlings at a low concentration of TIBA
White-green petals 0 2.5 5.3
supports this presumption (Fig. 5A). TIBA at a wide
Plant Physiol. Vol. 133, 2003 117
Al-Hammadi et al.

Figure 2. Comparison of the development of

embryo in poc mutant (A, C, E, G, and I) and
wild-type plants (B, D, F, H, and J). A and B,
Globular stage 10 DAP. C and D, Late globular
stage (13 DAP). E and F, Triangular/early heart
stage (15 DAP). G and H, Late heart/torpedo
stage (18 DAP). I and J, Magnification of medial
epidermal cells from the right-hand margin re-
gion of embryos shown in E and F, respectively.
Arrowheads, Cell boundaries in an epidermal
cell file. Scale bars: A to D, 10 ␮m; E to G, 20
␮m; and I and J, 5 ␮m.

range of concentrations tested (0.1–10 ␮m) brought mediated stimulation of elongation was seen for a
about a stimulation of hypocotyl elongation in the wide range of concentrations (0.1–10 ␮m).
mutant relative to the untreated control. At 0.3 ␮m The response to auxin was normal in roots of poc
concentration, TIBA promoted elongation of the poc seedlings as evidenced by similarity to wild type for
hypocotyls by 43%, compared with a minor 10% auxin-mediated inhibition of root elongation (Fig.
reduction in wild type (Fig. 5C). At 10 ␮m, hypocot- 5E). The inhibitory action of auxin also indicates that
yls were 18% longer than untreated seedlings in the reduced elongation of poc roots might be due to
case of the mutant, whereas wild type showed a 22% overaccumulation of auxin. The possibility that
reduction relative to the untreated control. shorter hypocotyl length of dark-grown mutant seed-
The root length of light-grown poc seedlings was lings resulted from auxin deficiency was examined
nearly 3 times less than the wild type (Fig. 5B), but by studying elongation of excised hypocotyls in the
root tip is normal as visualized by amyloplast stain-
presence of auxin. Although auxin only slightly stim-
ing (Benjamins et al., 2001). The possible relationship
ulated elongation of wild type, it significantly stim-
of the short root phenotype with enhanced PAT in
the poc mutant was examined by application of TIBA. ulated elongation of mutant hypocotyl (Fig. 5F), in-
The inclusion of 0.5 ␮m TIBA could fully restore the dicating a likely deficiency of auxin in the mutant
poc root length to a level similar to that of the wild tissue. Taken together, these data show that several
type. Interestingly, this low concentration of TIBA of the phenotypes of the poc mutant can be ascribed
had no significant effect on the length of wild type to a depletion of auxin arising from increased PAT
roots (Fig. 5D). For roots, as for hypocotyls, TIBA- and not to a reduced sensitivity to auxin because the
mutant retains substantial sensitivity to exogenously
applied auxin.
Examination of transverse sections of poc hypoco-
tyls revealed alteration in anatomy, particularly
with respect to vascular differentiation (Fig. 6). In
wild-type seedlings, the vascular bundles were usu-
ally arranged as four bundles (two pairs of two
bundles) with a central pith. A distinct feature of the
poc mutant was that vascular bundles were arranged
near each other due to a reduction in the central pith
region (Fig. 6C). Chemical inhibition of the PAT in
poc seedlings resulted in proliferation of xylem ves-
sels with appearance of central pith and arrange-
ment of bundles similar to untreated wild-type con-
trol (Fig. 6D). In contrast, in the presence of TIBA,
Figure 3. Scanning electron micrograph of epidermal cells of light-
grown seedlings. A and B, One-week-old cotyledons showing that
the proliferation of xylem vessels in wild type led to
the poc (B) has smaller cells. C and D, One-week-old hypocotyls fusion of adjacent bundles (Fig. 6B). These results
showing that poc (D) have shorter and less broad cells. Scale bars in indicate that changes in the vasculature of the poc
A to D: 50 ␮m. mutant could be due to altered levels of auxin.
118 Plant Physiol. Vol. 133, 2003
 Polar Auxin Transport in Tomato

Figure 4. Auxin polar transport in the presence or absence of TIBA. A, Auxin polar transport was measured using the method
of Okada et al. (1991) in stem sections of 5-week-old light-grown plants. The basal end of stem segments was submerged
in a solution containing [14C] IAA in an Eppendorf tube (Eppendorf Scientific, Westbury, NY) and after 4 h, a 5-mm section
from non-submerged end of segments was excised, and the amount of radioactivity was determined. Error bars ⫽ SE of five
replicates. B, Auxin polar transport was measured using the method of Daniel et al. (1989) in stem sections of 4-week-old
light-grown plants. The stem segments were sandwiched on glass microscopic slides between the receiver and donor blocks
of agar, and the setup was placed vertically in a humid chamber. After 4 h of incubation, the amount of radioactivity was
counted in the receiver blocks. Error bars ⫽ SE of five replicates. C, Auxin efflux rate was measured by the retention of the
amount of [14C] IAA in the hypocotyls of 3-week-old light grown plants. The cut segments of hypocotyls were floated on a
solution containing [14C] IAA either with or without TIBA. Then, segments were incubated for another 2 h in the same buffer
without IAA/TIBA and counted for radioactivity. Error bars ⫽ SE of five replicates. D, Absorption spectrum of flavonoids
extracted from stems of 4-week-old light-grown poc and wild-type plants.

Enhanced PAT Alters Gravitropic Curvatures that case, accumulation of auxin at the root tip due to
enhanced PAT in the mutant would be expected to
The experiments on the rescue of the poc phenotype
increase the amount of basipetal flow of auxin lead-
and auxin action on organ elongation indicated that
ing to higher curvatures. For poc seedlings, a higher
enhanced PAT may cause depletion of auxin in hy-
root gravitropic curvature was observed compared
pocotyl tissue and at the same time may cause over
with wild type (Fig. 7B). Interestingly, the gravitropic
accumulation of auxin at the root tip. Such alteration
curvature of roots of the poc mutant showed devia-
in auxin level is expected to affect physiological pro-
tion from wild type in two aspects in an entirely
cesses such as gravitropic curvature that are related
opposite way from that seen for the hypocotyl. First,
to auxin levels or distribution. Therefore, we com-
the lag period of curvature in poc was reduced to 10
pared gravitropic curvature of the hypocotyl and of
min compared with a lag of 15 min in wild type.
the root in the poc mutant with that in wild type. The
Second, for poc, a much higher degree of curvature
gravitropic curvature of the poc hypocotyl differed
was seen compared with wild type.
from wild type in two aspects (Fig. 7A). First, onset of
curvature in poc seedlings could be seen only after a
lag period of 1 h, which was almost twice that re- DISCUSSION
quired for wild type. Second, at all time points, the Phenotypes Exhibited by poc Mutant Are Most Likely
magnitude of curvature was much lower than that Caused by an Increase in Polar Transport of Auxin
for wild-type seedlings. In fact, some poc seedlings
showed only a little curvature even after 5 h of gra- The strong pleiotropic effect of the poc mutation
vistimulation. However, after 24 h, these poc seed- lasting throughout the plant life cycle likely signifies
lings showed significant curvature, albeit the extent modification in some central physiological processes
of the curvature in poc seedlings was less than in the regulating plant development. The observed devel-
wild type. opmental abnormalities in the poc mutant such as
It is believed that curvature of the root tip on increase in cotyledon numbers, change in shape and
gravistimulation results from basipetal flow of auxin size of the leaf, loss of lobing in the leaflet, ectopic
from the root tip into cortical and epidermal cells. In formation of organs on the rachis, increased number
Plant Physiol. Vol. 133, 2003 119
Al-Hammadi et al.

tensive branching of the inflorescence with numer-

ous abnormal flowers (Fig. 1).
Several lines of evidence indicate that developmen-
tally regulated auxin levels and distribution specify
vascular differentiation during normal plant organo-
genesis (Ye, 2002). Such a role for auxin as an inducer
of vascular differentiation is seen in auxin-
overproducing transgenic plants, wherein an excess
of auxin levels causes increase in the amounts of
vascular tissue (Klee et al., 1987). PAT is essential for
the formation of spatially organized patterns of vas-
cular tissue, and a reduction of PAT in ifl1 and pin1
mutants is accompanied by proliferation of xylem in
the vascular bundles of inflorescence stems (Gäl-
weiler et al., 1998; Mattsson et al., 1999; Tsiantis et al.,
1999; Zhong and Ye, 2001). Because reduction in PAT
increases vascular proliferation, increase in PAT is
expected to reduce auxin levels in the stem and dis-
play the opposite effect. In fact, auxin-deficient trans-
genic plants show a reduction in vascular develop-
ment (Romano et al., 1991). The anatomical
alterations observed in poc, such as smaller epidermal
cells and compact placement of vascular bundles
with reduction of the central pith (Fig. 6), are in
conformity with the view that these changes may
reflect reduced auxin levels due to increase in PAT.
Figure 5. Hypocotyl and root growth of seedlings in the presence of One way to ascertain a causal connection between
TIBA and IAA. A, Morphology of 9-d-old dark-grown wild-type (a the mutation and a physiological process is to phe-
and c) and poc (b and d) seedlings in the presence (c and d) or nocopy the mutation by external application of bio-
absence (a and b) of TIBA (0.5 ␮M). B, Morphology of 9-d-old
active molecules. In several instances, phenotypes of
light-grown wild-type (a and c) and poc (b and d) seedlings in the
presence (c and d) or absence (a and b) of TIBA (0.5 ␮M). C, Effect of
mutants defective in PAT have been phenocopied in
various concentrations of TIBA on elongation of hypocotyl length of wild type by application of PAT inhibitors (Okada et
10-d-old dark-grown poc seedlings compared with the wild type. D, al., 1991; Gälweiler et al., 1998). Although a lack of
Effect of various concentrations of TIBA on elongation of root lengths agonists that can stimulate PAT in plants precludes
of 9-d-old light-grown poc seedlings compared with wild type. E, phenocopy of the poc mutation in wild type, we
Effect of various concentrations of IAA on root elongation of 9-d-old tested whether the application of PAT inhibitors to
light-grown wild-type seedlings compared with poc. F, Effect of IAA the poc mutant could rescue the mutant phenotype to
in promoting elongation of hypocotyl segments of 5-d-old dark- wild type. Etiolated seedlings of the poc mutant char-
grown poc seedlings compared with wild type.
acteristically show a shorter hypocotyl, whereas the
light-grown mutant seedlings exhibit a shorter pri-
mary root than the wild type. A rescue of the phe-
of flowers in the inflorescence, and fusion of floral notype is clearly seen in seedlings raised in the pres-
organs are broadly reminiscent of alteration in phy- ence of TIBA (Fig. 5). TIBA increases the root and
tohormone action. Examination of mutant seedlings hypocotyl lengths of the mutant seedlings to close to
revealed a 3-fold higher level of PAT. During that of the wild type. A similar rescue of phenotype
postembryonic development, interference with PAT by TIBA was also seen for anatomical alteration in
by mutation or exogenous application of PAT inhib- the poc mutant. Taken together, we can reasonably
itors results in defects in leaf morphology and vena- speculate that the aberrant morphology of the poc
tion, inflorescence architecture, vascular anatomy, mutant may be induced by an enhancement in PAT.
and tropic movement of plants (Friml and Palme, Several lines of evidence support the assumption
2002). Interestingly, several of the morphological and concerning elevation in the rate of auxin transport in
anatomical changes in the poc mutant are the reverse the poc mutants. First, direct quantification of basipe-
of those observed for Arabidopsis mutants such as tal transport shows that the rate of auxin transport in
pin1 (Okada et al., 1991; Gälweiler et al., 1998), tir3 the poc mutant is nearly 3-fold higher than in the wild
(Ruegger et al., 1997), and ifl1 (Zhong and Ye, 2001) type, and the exogenous application of TIBA reduces
that display a reduction in PAT. The stronger alleles the transport rate close to the wild-type control. Sec-
of these mutants develop a pin-shaped inflorescence ond, tomato stems preloaded with radiolabeled
devoid of floral primordia and abnormal leaves. In auxin show a higher rate of auxin efflux in the poc
contrast, the poc mutant shows features such as ex- mutant compared with wild type. Again, in this case,
120 Plant Physiol. Vol. 133, 2003
 Polar Auxin Transport in Tomato

Figure 6. Transverse sections showing the vas-

culature in hypocotyls of 15-d-old light-grown
poc seedlings in the presence or absence of
TIBA (10 ␮M). Sections were taken from the
middle portion of hypocotyls and stained with
safranine. A, Wild type; B, wild type ⫹ TIBA; C,
poc mutant; D, poc mutant ⫹ TIBA. Note the
difference in placement of the vascular bundles
between mutant and wild type. Scale bars in A
to D: 50 ␮m.

TIBA reduces the efflux rate in the mutant to the 2002b). The observed delay in onset and reduction in
levels observed for wild type. The auxin transport in magnitude of gravitropic curvature in poc hypocotyls
inflorescence stems and hypocotyls of the flavonoid- is consistent with reduction in auxin levels in the
deficient tt4 mutant is elevated over wild type by tissue due to enhanced PAT. Reduced lateral flow of
about 2-fold (Brown et al., 2001). The observed in- auxin also correlates with a reduction in the width of
crease in PAT in the poc mutant cannot be explained epidermal cells in poc hypocotyls. The paucity of
by a general reduction in the levels of flavonoids auxin in hypocotyls is also indicated by greater stim-
because the mutant plants had nearly the same level ulation of hypocotyl elongation by auxin in the poc
of flavonoids as in the wild type. Together, these data mutant than wild type and is consistent with its
strengthen the view that the poc mutation enhances depletion due to increased PAT.
the rate of auxin transport, and it acts at a step other One of the obvious consequences of enhanced PAT
than flavonoid biosynthesis. is the likelihood of channelization of most auxin to
the root pole, causing its accumulation at the root tip.
Enhanced Auxin Polar Transport Likely Alters Auxin Three lines of evidence point to such an overaccumu-
Distribution Pattern in the poc Mutant lation in the poc mutant based on current physiolog-
ical models of auxin-mediated root growth and
Auxin distribution has been implicated as a key tropic curvature (Sabatini et al., 1999; Masson et al.,
regulator of several developmental processes such as 2002). First, a reduction in the root tip growth ob-
cell elongation and tropic curvatures. Mutations af- served is consistent with an increased level of auxin
fecting PAT in several instances may elicit their phe- because beyond a threshold level, auxin acts as an
notype by consequent disturbance of the auxin bal- inhibitor of growth. In fact, such accumulation of
ance of the tissues (Sabatini et al., 1999). Etiolated auxin at the root tip in transgenic pinoid-
hypocotyls of mdr1 mutants, defective in PAT, dis- overproducing plants leads to total collapse of the
play larger gravitropic and phototropic curvatures root meristem, and this could be rescued by applica-
probably due to accumulation of auxin (Spalding et tion of PAT inhibitors (Benjamins et al., 2001). An
al., 2002). On the contrary, increase in PAT may increase in root length of poc seedlings by application
result in faster removal of auxin, reducing its lateral of PAT inhibitors indicates that the increased accu-
flow. One critical physiological assay for the lateral mulation of auxin may be responsible for the de-
flow of auxin is the magnitude of gravitropic curva- creased root growth. Second, adult poc plants pro-
ture that is related to its lateral distribution as per duce more lateral roots than the wild type (Reed et
Cholodny and Went’s hypothesis (Trewavas, 1992). al., 1998). Third, an increased accumulation of auxin
The decrease in the lateral flow of auxin in the pin3 at the root meristem would also increase the “back
mutant also correlates with a reduction in the tropic flow” of auxin in the basipetal direction in epidermal
curvature of Arabidopsis seedlings (Friml et al., and cortical cells of the root. Such increase in the
Plant Physiol. Vol. 133, 2003 121
Al-Hammadi et al.

determination of pattern specificity (Souter and

Lindsey, 2000; Berleth and Chatfield, 2002). Recent
reports have brought some support to the role of
auxin as a “morphogen” regulating cell differentia-
tion in a concentration-dependent manner (Sabatini
et al., 1999; Friml et al., 2002a). Current research on
embryo development in Arabidopsis mutants has
highlighted that the transition from the globular to
the heart stage requires elements participating in
auxin perception and distribution. A role for auxin in
the progression of embryogenesis is indicated by the
observation that a null mutation in the auxin-binding
protein ABP1 arrests Arabidopsis embryogenesis at
the globular stage (Chen et al., 2001). Similarly, fail-
ure to establish a properly localized distribution of
auxin transporting proteins during the globular stage
in the gnom mutant results in an abnormal embryo
devoid of cotyledonary primordia (Mayer et al., 1993;
Steinmann et al., 1999).
The appearance of cotyledonary poles on a devel-
oping embryo marks the end of the globular stage
leading to establishment of bilateral symmetry. Avail-
able evidence indicates that development of the auxin
transport system during the globular stage is related
to the subsequent appearance of cotyledon primordia.
In a range of plant species, disruption of PAT either by
a mutation such as pin1 (Liu et al., 1993) or gnom
(Mayer et al., 1993) or by using inhibitors of auxin
transport during zygotic and also somatic embryogen-
esis (Hadfi et al., 1998; Liu et al., 1993) disrupts em-
bryonic development with generation of embryos
with fused or missing cotyledons. Thus, normal auxin
Figure 7. Analysis of gravitropic response in seedlings. A, Kinetics of transport appears to be one of the prerequisites for the
gravitropic response in the hypocotyls of 7-d-old light-grown wild- radial globular embryo to progress to the bilaterally
type and poc seedlings. Seedlings grown in plastic cuvettes were symmetrical heart stage embryo (Liu et al., 1993; Hadfi
reoriented 90° relative to the gravity vector. The curvature was et al., 1998). However, our knowledge about the
determined by photographing the seedlings at the given time inter- events leading to onset of cotyledon initiation during
vals using a PC camera. B, Gravitropic response in the roots of embryogenesis is limited. Embryonic fate mapping
1-d-old light-grown wild-type and poc seedlings. Seedlings grown on using chimeric tissue indicated that in Arabidopsis,
vertical agar plates were reoriented 90° relative to the gravity vector.
first one cotyledon gets determined and that in turn
The curvature was determined by photographing the roots at the
given time intervals using a PC camera. directs the positioning of the other cotyledon at the
opposite end. It is suggested that some kind of lateral
inhibition is responsible for this process, directing the
basipetal flow of auxin in the root, as per the “foun- formation of new primordia at the site of lowest inhi-
tain model” of auxin-mediated root gravitropism, bition (Woodrick et al., 2000).
would be predicted to stimulate the gravitropic root The association of defects in auxin transport with
tip curvature in the poc mutant. Analysis of root alterations in cotyledon initiation/separation in a
gravitropic curvature broadly substantiates this view number of instance points to the likelihood of auxin
because gravistimulated poc root tips showed a much playing an important role in the initiation of cotyle-
shorter lag period and nearly twice the curvature don development. Based on reports that indicate as-
compared with wild type. sociation between auxin transport and cotyledon de-
velopment, it seems possible that faster removal of
auxin may result in initiation of additional cotyle-
Is Polar Transport of Auxin Related to Initiation of
Cotyledons in the Globular Embryo?
dons. In fact, based on their results on auxin and PAT
inhibitor application to developing mustard em-
Auxin is considered as a principal regulator of bryos, Hadfi et al. (1998) proposed that polar trans-
patterning during embryogenesis. Because auxin is port removes auxin from the area between the two
polarly transported and may act in a non-cell auton- emerging cotyledon primordia causing separation of
omous fashion, it can provide the necessary input for cotyledons. Therefore, the removal of auxin by en-
122 Plant Physiol. Vol. 133, 2003
 Polar Auxin Transport in Tomato

hanced polar transport could deplete auxin below a Mutants Screening and Genetic Analysis
threshold level in cells of the apical dome, leading to The ethyl methanesulfonate (EMS) mutagenesis was performed essen-
initiation of additional cotyledon primordia. The ap- tially following the procedure of Koornneef et al. (1990). Approximately
pearance of multiple cotyledons in poc embryos at the 5,000 seeds of tomato cv Ailsa Craig were soaked in a solution of 60 mm
transition from globular to heart stage is indicative of EMS for 24 h in the dark at room temperature. Thereafter, the seeds were
washed with distilled water, and M1 generation plants were grown in the
such a link between auxin transport and cotyledon field. From fruits of a population of approximately 1,500 M1 plants, M2
formation. Likewise, the reduction in size of epider- seeds were harvested in bulk. A population of approximately 50,000 EMS-
mal cells in poc embryos compared with wild type mutagenized M2 seedlings was screened for putative cotyledon mutants.
One-week-old seedlings were screened for altered cotyledon number and
could be a consequence of increased PAT.
size. Of 25 putative mutants showing multiple cotyledons, only nine mu-
Though an association of increase in cotyledon tants survived. Because the mutants were male sterile, these were rescued
number with increase in PAT is seen for the poc by manually pollinating with wild-type pollens, and F1 heterozygous seeds
mutant of tomato, it is at variance with the pinoid were obtained. Subsequently, mutants were also manually self-pollinated,
and homozygous seeds were obtained. The complementation analysis using
mutant of Arabidopsis, which shows polycot seed- isolated mutant lines indicated that all nine mutants were allelic. For all
lings but reduced PAT in inflorescence stems (Ben- experiments, seeds multiplied from the mutant line poc1-1 that was back-
nett et al., 1995). A recent report suggested that PI- crossed twice were used.
NOID acts as a positive regulator of PAT (Benjamins
et al., 2001), whereas our results indicate POC to be a
negative regulator. It is proposed that PINOID may Embryogenesis
be required for proper positioning of cotyledonary Both the wild-type and poc mutant flowers were emasculated and man-
primordia (Benjamins et al., 2001), whereas POC ac- ually self-pollinated. The developing fruits were harvested at regular inter-
vals starting 10 DAP. Pistils were removed from the developing fruits and
tion might be related to separation of cotyledon pri-
fixed in ethanol:acetic acid (6:1 [v/v]) overnight. After several washes in
mordia (Hadfi et al., 1998). In such a case, these two 100% ethanol followed by washing in 70% ethanol, the pistils were pre-
might affect different physiological processes and served in 70% ethanol. The pistils were then cleared in a mixture of chloral
still show similar phenotypic effects. It could be hydrate:glycerol:water (8:1:2 [w/v]) overnight (Berleth and Jurgens, 1993)
and dissected under a dissection microscope to reveal the ovules, which
equally plausible that enhanced PAT in Arabidopsis
were then further dissected to remove embryos. The embryos were observed
is not related to development of polycotyly. For ex- using a Zeiss Axiophot microscope under DIC optics (Zeiss, Oberkochen,
ample, the Arabidopsis tt4 mutant shows enhanced Germany) and photographed using black and white film (15-25 ASA; Sid-
PAT but does not exhibit a polycot phenotype diqi et al., 2000).
(Brown et al., 2001).
In summary, our results indicate that the poc mu-
PAT Assay
tation of tomato shows increased PAT and that this
could be responsible for its pleiotropic phenotype. The polar transport of auxin was assayed in the stem segments of
We have located the poc gene on chromosome nine of light-grown plants using two different protocols outlined by Okada et al.
(1991) and Daniel et al. (1989) with some modifications. The height and the
tomato (M. Kavitha, P. Bauer, M.S. Sharada, A.S.A. diameters of the stems of both poc and wild-type plants were nearly similar.
Al-Hammadi, P. Janila, S. Negi, R. Sharma, unpub- For the Okada et al. (1991) protocol, 10 stem segments (2.5 cm) were cut 2
lished data) and are currently fine mapping it with an mm below the cotyledonary node from 5-week-old light-grown plants. The
segments were first floated for 2 h in 5 mm phosphate buffer (pH 5.8)
aim to isolate and determine its role in PAT.
containing either 1 ␮m IAA or 20 ␮m TIBA. To avoid a gravitropic response
during that period, this pre-incubation took place on a shaker. Thereafter,
either the apical or basal end of the segments (for basipetal or acropetal
transport, respectively) was submerged in 50 ␮L of 5 mm phosphate buffer
MATERIALS AND METHODS (pH 5.8) with 27 nCi mL⫺1 (specific activity ⫽ 4.2 mCi mmol⫺1 of [1,2-14C]
IAA; BRIT, Mumbai, India) and 1 ␮m IAA (unlabeled) in a 1.5-mL Eppen-
Plant Growth Conditions
dorf tube. After 4 h, a 5-mm section from the non-submerged end of
Tomato (Lycopersicon esculentum L. cv Ailsa Craig) seeds were surface segments was excised and floated overnight in 3 mL of Bayer’s solution. The
sterilized and sown on filter papers. After emergence of the radicle, seed- radioactivity was counted in a Beckman liquid scintillation counter. Similar
lings were grown on vermiculite either under continuous white light (100 to Brown et al. (2001), we also found that a 4-h transport period was
sufficient to measure PAT in tomato, unlike 18 h used by Okada et al. (1991).
␮mol m⫺2 s⫺1) or in darkness at 25°C. Unless otherwise mentioned, for all
For the Daniel et al. (1989) protocol using agar blocks, stem segments (1.2
experiments the plant age was counted from the time point of emergence of
cm) of 4-week-old tomato plants were excised 2 mm below the cotyledonary
the radicle. To test the viability, pollens were germinated on Brewbaker and
node and incubated for 2 h in 5 mm phosphate buffer (pH 5.8) containing
Kwack’s media ( Brewbaker and Kwack, 1963), and the percentage pollen
either 1 ␮m IAA or 20 ␮m TIBA on a shaker. These segments were sand-
germination was calculated after staining with 1% (w/v) acetocarmine. Leaf
wiched on glass microscopic slides between receiver (1.5% [w/v] agar in
and cotyledon venation was examined by depigmenting tissues in acidified water, 2.5 ⫻ 0.4 ⫻ 0.4 cm) and donor blocks (1.5% [w/v] agar in 5 mm
methanol (2% [v/v] HCl) followed by clearing in a mixture of chloral phosphate buffer [pH 5.8] containing 1 ␮m IAA and approximately 17 nCi
hydrate:glycerol:water (8:1:2 [w/v]) and staining with p-rosaniline hydro- mL⫺1 [14C-IAA], 2.5 ⫻ 0.8 ⫻ 0.4 cm). Twelve segments for each of the donor
chloride. Flavonoid content of 4-week-old light-grown plants (n ⫽ 10, three receiver block units were placed either in basipetal (apical end toward
replicates) was estimated according to Harborne (1967). The shoots of these donor block) or in acropetal direction (with basal end toward donor). The
plants were excised and extracted in 10 mL of acidified (1% [v/v] HCl) donor receiver block units were placed vertically in a humid chamber at
methanol for 24 h in darkness with shaking. After centrifugation, the ab- 25°C ⫾ 2°C, and the system was inverted to prevent drainage of [14C IAA]
sorption spectra were recorded. The total flavonoid level is expressed as onto the receiver blocks. After 4 h of incubation, the receiver blocks were
A330/shoot. The experimental data for the cell size and morphological removed and placed in 3 mL of liquid scintillation cocktail. The samples
variation was also analyzed by a two-tailed Student’s t test, and P values were shaken at 100 rpm for 2 h and left overnight at 25°C ⫾ 2°C before
were calculated. counting in a scintillation counter.

Plant Physiol. Vol. 133, 2003 123

Al-Hammadi et al.

Auxin Efflux Assay For root gravitropism, seeds with just emerged radicles were sown on
1.7% (w/v) agar in petri plates, and plates were kept vertically under
Auxin efflux in the hypocotyls was assayed according to the procedure of continuous white light. After 24 h, when roots elongated to 5 to 6 mm, the
Bernasconi (1996). Twelve hypocotyls of 3-week-old light-grown seedlings plates were rotated by 90°, and images were recorded at every 5 min for 2 h
were cut into segments (4 mm). The segments were floated with shaking for using an Ezonics PC camera. Each petri plate had three poc and three
2 h in 5 mm phosphate buffer (pH 5.8) with 1% (w/v) Suc containing 0.1 ␮Ci wild-type seedlings, and the experiment was repeated about 10 times.
mL⫺1 [14C] IAA either with or without 20 ␮m TIBA. Then, the segments
were rinsed and incubated for another 2 h in the same buffer without
IAA/TIBA. After a final rinse, the segments were placed in Bayer’s solution ACKNOWLEDGMENTS
and counted for radioactivity as described above.
We wish to thank Dr. Bernie Carroll (University of Queensland, Austra-
lia) for providing us the dem mutant seeds, Dr. Shashi Singh (Centre for
Effect of TIBA on Organ Elongation Cellular and Molecular Biology, Hyderabad, India) for light microscopy,
and Dr. Manjunath (Central Instruments Laboratory, University of Hyder-
Surface-sterilized seeds of tomato were germinated in the dark. After the abad, Hyderabad, India) for SEM.
emergence of the radicle, seeds were transferred either onto petri plates or
germination boxes containing 1.7% (w/v) agar support prepared with one- Received April 15, 2003; returned for revision May 18, 2003; accepted May
tenth-strength Murashige and Skoog media (inorganic salts only, Murashige 24, 2003.
and Skoog, 1962). For studying the effect of TIBA on root length, seeds were
sown on agar containing different concentrations of TIBA in the range of 0
to 20 ␮m. The petri plates were vertically oriented after 9-d seedlings were LITERATURE CITED
removed, and the root lengths were measured. To study the effect of TIBA
Benjamins R, Quint A, Weijers D, Hooykaas P, Offringa R (2001) The
on hypocotyl length, seeds were sown on 1.7% (w/v) agar in transparent
PINOID protein kinase regulates organ development in Arabidopsis by
germination boxes containing different concentrations of TIBA in the range
enhancing polar auxin transport. Development 128: 4057–4067
of 0 to 20 ␮m. The germination boxes were kept in the dark for 10 d. On the
Bennett MJ, Marchant A, Green HG, May ST, Ward SP, Millner PA,
10th d, the hypocotyl lengths were measured.
Walker AR, Schulz B, Feldmann KA (1996) Arabidopsis AUX1 gene: a
permease like regulator of root gravitropism. Science 273: 948–950
Bennett SRM, Alvarez J, Bossinger G, Smyth DR (1995) Morphogenesis in
Effect of Auxin on Organ Elongation pinoid mutants of Arabidopsis thaliana. Plant J 8: 505–520
Berleth T, Chatfield S (2002) Embryogenesis: pattern formation from a
The method of estimating elongation of hypocotyls was essentially sim-
single cell. In CR Somerville, EM Meyerowitz, eds, The Arabidopsis
ilar to that described earlier by Kelly and Bradford (1986), with the variation
Book. American Society of Plant Biologists, Rockville, MD,
that the hypocotyls were used immediately after excision and were not
http://www.aspb.org/downlaods/arabidopsis/berleth.pdf
depleted of endogenous auxin. One-centimeter segments of hypocotyls
Berleth T, Jurgens G (1993) The role of the monopteros gene in organizing
were excised from 5-d-old dark-grown seedlings and floated in 5 mm
the basal body region of the Arabidopsis embryo. Development 118:
phosphate buffer (pH 5.8) containing 1% (w/v) Suc with and without 1 ␮m
575–587
IAA. The length of each segment was recorded after 4 h. For study on root
Bernasconi P (1996) Effect of synthetic and natural protein tyrosine kinase
length, seedlings were grown on agar with or without IAA in the light on
inhibitors on auxin efflux in zucchini (Cucurbita pepo) hypocotyls. Physiol
vertical petri plates, and the root lengths were measured after 9 d.
Plant 96: 205–210
Bernasconi P, Bhavesh CP, Reagan JD, Subramanian MV (1996) The
N-1-naphthylphthalamic acid-binding protein is an integral membrane
SEM
protein. Plant Physiol 111: 427–432
The middle region from hypocotyls and cotyledons of 7-d-old light- Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen
grown seedlings was excised and mounted on stubs using a double-sided germination and pollen tube growth. Am J Bot 50: 859–865
adhesive tape. In the case of cotyledons, the abaxial side was examined for Brown DE, Rashotte AM, Murphy AS, Normanly J, Tague BW, Peer WA,
epidermal cell shape. The stubs were plunged in liquid nitrogen, and the Taiz L, Muday GK (2001) Flavonoids act as negative regulators of auxin
frozen organs were immediately examined in a environmental scanning transport in vivo in Arabidopsis. Plant Physiol 126: 524–535
electron microscope (Philips, Eindhoven, The Netherlands). Chen J-G, Ullah H, Young JC, Sussman MR, Jones AM (2001)ABP1 is
required for organized cell elongation and division in Arabidopsis embry-
ogenesis. Genes Dev 15: 902–911
Light Microscopy Chen R, Hilson P, Sedbrook J, Rosen E, Caspar T, Masson PH (1998) The
Arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the
Free-hand sections were cut from the hypocotyls of 15-d-old light-grown polar-auxin-transport efflux carrier. Proc Natl Acad Sci USA 95:
seedlings. At this age, hypocotyls were approximately 3.5 cm long. The 15112–15117
sections were cut from at least 10 seedlings from the central region of Christensen SK, Dagenais N, Chory J, Weigel D (2000) Regulation of auxin
hypocotyls at a distance of 1.5 to 2 cm from the cotyledonary node. The cut response by the protein kinase PINOID. Cell 100: 469–478
sections were immediately stained in safranine and were destained in water. Coenen C, Christian M, Lüthen H, Lomax TL (2003) Cytokinin inhibits a
The sections were mounted on slides in water and were photographed in a subset of diageotropica-dependent primary auxin responses in tomato.
Zeiss Axiophot microscope using DIC optics. Plant Physiol 131: 1692–1704
Daniel SG, Rayle DL, Cleland RE (1989) Auxin physiology of tomato
mutant diageotropica. Plant Physiol 91: 804–807
Analysis of Gravitropism Friml J, Palme K (2002) Polar auxin transport-old questions new concepts?
Plant Mol Biol 49: 273–284
Gravity response in the hypocotyls was measured using 7-d-old light- Friml J, Benková E, Blilou I, Wisniewska J, Hamann T, Ljung K, Woody S,
grown seedlings grown vertically in plastic cuvettes filled with vermiculite. Sandberg G, Scheres B, Jürgens G et al. (2002a) AtPIN4 mediates
At the time of gravistimulation, seedlings were turned 90°, and the cuvettes sink-driven auxin gradients and root patterning in Arabidopsis. Cell 108:
were arranged in a rack. Seedlings were photographed using an Ezonics 661–673
(Ezcam) PC camera (Ezonics Corporation, Pleasanton, CA) at every 15-min Friml J, Wisniewska J, Benková E, Mendgen K, Palme K (2002b) Lateral
interval initially and later at every 30 min for 6 h. For each experiment, five relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis.
poc and five wild-type seedlings were used and the experiment was re- Nature 415: 806–809
peated six to eight times. The angles were measured using Image Tool Gälweiler L, Guan C, Müller A, Wisman E, Mendgen K, Yephremov A,
program after subtracting the zero time point image from the image at Palme K (1998) Regulation of polar auxin transport by AtPIN1 in Arabi-
required time points. dopsis vascular tissue. Science 282: 2226–2230

124 Plant Physiol. Vol. 133, 2003

 Polar Auxin Transport in Tomato

Geldner N, Anders N, Wolters H, Keicher J, Kornberger W, Muller P, Müller A, Guan C, Gälweiler L, Tanzler P, Huijser P, Marchant A, Parry
Delbarre A, Ueda T, Nakano A, Jurgens G (2003) The Arabidopsis G, Bennett M, Wisman E, Palme K (1998) AtPIN2 defines a locus of
GNOM ARF-GEF mediates endosomal recycling, auxin transport, and Arabidopsis for root gravitropism control. EMBO J 17: 6903–6911
auxin-dependent plant growth. Cell 112: 219–230 Murashige T, Skoog F (1962) A revised medium for rapid growth and
Geldner N, Friml J, Stierhof Y-D, Jürgens G, Palme K (2001) Auxin bioassays with tobacco tissue culture. Physiol Plant 15: 493–497
transport inhibitors block PIN1 cycling and vesicle trafficking. Nature Murphy A, Peer WA, Taiz L (2000) Regulation of auxin transport by
413: 425–428 aminopeptidases and endogenous flavonoids. Planta 211: 315–324
Gil P, Dewey E, Friml J, Zhao Y, Snowden KC, Putterill J, Palme K, Estelle Okada K, Ueda J, Komaki MK, Bell CJ, Shimura Y (1991) Requirement of
M, Chory J (2001) BIG: a calossin-like protein required for polar auxin the auxin polar transport system in early stages of Arabidopsis floral bud
transport in Arabidopsis. Genes Dev 15: 1985–1997 formation. Plant Cell 3: 677–684
Hadfi K, Speth V, Neuhaus G (1998) Auxin-induced developmental pat- Pickett FB, Wilson AK, Estelle M (1990) The aux1 mutation of Arabidopsis
terns in Brassica juncea embryos. Development 125: 879–887 confers both auxin and ethylene resistance. Plant Physiol 94: 1462–1466
Harborne JB (1967) Comparative Biochemistry of Flavonoids. Academic Rashotte AM, DeLong A, Muday GK (2001) Genetic and chemical reduc-
Press, London tions in protein phosphatase activity alter auxin transport gravity re-
Jacobs M, Gilbert SF (1983) Basal localization of the presumptive auxin sponse and lateral root growth. Plant Cell 13: 1683–1697
transport carrier in pea stem cells. Science 220: 1297–1300 Raven J (1975) Transport of indoleacetic acid in plant cells in relation to pH
Jacobs M, Rubery PH (1988) Naturally occurring auxin transport regulators. and electric potential gradients, and its significance for polar IAA trans-
Science 241: 346–349 port. New Phytol 74: 163–172
Keddie JS, Carroll BJ, Thomas CM, Reyes MEC, Klimyuk V, Holtan H, Reed RC, Brady SR, Muday GK (1998) Inhibition of auxin movement from
Gruissem W, Jones JDG (1998) Transposon tagging of the defective the shoot into the root inhibits lateral root development in Arabidopsis.
embryo and meristems gene of tomato. Plant Cell 10: 877–888 Plant Physiol 118: 1369–1378
Kelly MO, Bradford KJ (1986) Insensitivity of the diageotropica tomato Romano CP, Hein MB, Klee HJ (1991) Inactivation of auxin in tobacco
mutant to auxin. Plant Physiol 82: 713–717
transformed with the indole acetic acid-lysine synthetase gene of Pseudo-
Klee HJ, Horsch RB, Hinchee MA, Hein MB, Hoffmann NL (1987) The
monas savastanoi. Genes Dev 5: 438–446
effects of overproduction of two Agrobacterium tumefaciens T-DNA auxin
Rubery PH, Sheldrake AR (1974) Carrier mediated auxin transport. Planta
biosynthetic gene products in transgenic petunia plants. Genes Dev 1:
118: 101–121
86–96
Ruegger M, Dewey E, Hobbie L, Brown D, Bernasconi P, Turner J, Muday
Koornneef M, Bosma TDG, Hanhart CJ, Van der Veen JH, Zeevaart JAD
G, Estelle M (1997) Reduced naphthylphthalamic acid binding in the tir3
(1990) The isolation and characterization of gibberellin-deficient mutants
mutant of Arabidopsis is associated with a reduction in polar auxin
in tomato. Theor Appl Genet 80: 852–857
transport and diverse morphological defects. Plant Cell 9: 745–757
Liu C-M, Xu Z-H, Chua N-H (1993) Auxin polar transport is essential for the
Sabatini S, Beis D, Wolkenfelt H, Murfett J, Guilfoyle T, Malamy J,
establishment of bilateral symmetry during early plant embryogenesis.
Benfey P, Leyser O, Bechtold N, Weisbeek P et al. (1999) An auxin-
Plant Cell 5: 621–630
dependent distal organizer of pattern and polarity in the Arabidopsis root.
Lomax TL, Muday GK, Rubery PH (1995) Auxin transport. In PJ Davies, ed,
Cell 99: 463–472
Plant Hormones, Physiology, Biochemistry and Molecular Biology. Klu-
Siddiqi I, Ganesh G, Grossniklaus U, Subbaiah V (2000) The dyad gene is
wer Academic Publishers, Dordrecht, The Netherlands, pp 509–530
Luschnig C, Gaxiola RA, Grisafi P (1998) Fink GR EIR1 a root-specific required for progression through female meiosis in Arabidopsis. Devel-
protein involved in auxin transport is required for gravitropism in Ara- opment 127: 197–207
bidopsis thaliana. Genes Dev 12: 2175–2187 Souter M, Lindsey K (2000) Polarity and signalling in plant embryogenesis.
Marchant A, Kargul J, May ST, Muller P, Delbarre A, Perrot-Rechenmann J Exp Bot 51: 971–983
C, Bennett MJ (1999) AUX1 regulates root gravitropism in Arabidopsis by Spalding E, Murphy A, Noh B (2002) Larger and faster tropisms in mdr
facilitating auxin uptake within root apical tissues. EMBO J 18: 2066–2073 mutants lacking polar auxin transport. American Society of Plant Biolo-
Masson PH, Tasaka M, Morita MT, Guan C, Chen R, Boonsirichai K (2002) gists, Minisymposium: Tropism. Abs #28003. http://abstracts.aspb.
Arabidopsis thaliana: a model for the study of root and shoot gravitropism. org/pb2002/public/M16/1053.html
In CR Somerville, EM Meyerowitz, eds, The Arabidopsis Book. American Steinmann T, Geldner N, Grebe M, Mangold S, Jackson CL, Paris S,
Society of Plant Biologists, Rockville, MD, http://www.aspb.org/down- Gälweiler L, Palme K, Jurgens G (1999) Coordinated polar localization
loads/arabidopsis/masson.pdf of auxin efflux carrier PIN1 by GNOM ARF GEF. Science 286: 316–318
Mattsson J, Sung ZR, Berleth T (1999) Responses of plant vascular systems Trewavas AJ (1992) FORUM: what remains of the Cholodny-Went Theory?
to auxin transport inhibition. Development 126: 2979–2991 Plant Cell Environ 15: 759–794
Mayer U, Buettner G, Jurgens G (1993) Apical-basal pattern formation in Tsiantis M, Brown MIN, Skibinski G, Langdale JA (1999) Disruption of
the Arabidopsis embryo: studies on the role of the gnom gene. Develop- auxin transport is associated with aberrant leaf development in maize.
ment 117: 149–162 Plant Physiol 121: 1163–1168
Muday GK, Brunn SA, Haworth P, Subramanian M (1993) Evidence for a Woodrick R, Martin PR, Birman I, Pickett FB (2000) The Arabidopsis em-
single naphthylphthalamic acid binding site on the zucchini plasma bryonic shoot fate map. Development 127: 813–820
membrane. Plant Physiol 103: 449–456 Ye Z-H (2002) Vascular tissue differentiation and pattern formation in
Muday GK, Lomax TL, Rayle DL (1995) Characterization of the growth and plants. Annu Rev Plant Biol 53: 183–202
auxin physiology of roots of the tomato mutant diageotropica. Planta 195: Zhong R, Ye Z-H (2001) Alteration of auxin polar transport in the Arabi-
548–553 dopsis ifl1 mutants. Plant Physiol 126: 549–563

Plant Physiol. Vol. 133, 2003 125