Alkamides Isolated from Plants Promote Growth and

Alter Root Development in Arabidopsis1

Enrique Ramı́rez-Chávez2, José López-Bucio2, Luis Herrera-Estrella*, and Jorge Molina-Torres

Departamento de Biotecnologı́a y Bioquı́mica (E.R.-C., J.M.-T.) and Departamento de Ingenierı́a Genética
(J.L.-B., L.H.-E.), Unidad Irapuato del Centro de Investigación y Estudios Avanzados del Instituto Politécnico
Nacional, Apartado Postal 629, 36500 Irapuato, Guanajuato, México

To date, several classes of hormones have been described that influence plant development, including auxins, cytokinins,
ethylene, and, more recently, brassinosteroids. However, it is known that many fungal and bacterial species produce
substances that alter plant growth that, if naturally present in plants, might represent novel classes of plant growth
regulators. Alkamides are metabolites widely distributed in plants with a broad range of biological activities. In this work,
we investigated the effects of affinin, an alkamide naturally occurring in plants, and its derivates, N-isobutyl-2E-decenamide
and N-isobutyl-decanamide, on plant growth and early root development in Arabidopsis. We found that treatments with
affinin in the range of 10⫺6 to 10⫺4 m alter shoot and root biomass production. This effect correlated with alteration on
primary root growth, lateral root formation, and root hair elongation. Low concentrations of affinin (7 ⫻ 10⫺6–2.8 ⫻ 10⫺5
m) enhanced primary root growth and root hair elongation, whereas higher concentrations inhibited primary root growth
that related with a reduction in cell proliferating activity and cell elongation. N-isobutyl-2E-decenamide and N-isobutyl-
decanamide were found to stimulate root hair elongation at concentrations between 10⫺8 to 10⫺7 m. Although the effects of
alkamides were similar to those produced by auxins on root growth and cell parameters, the ability of the root system to
respond to affinin was found to be independent of auxin signaling. Our results suggest that alkamides may represent a new
group of plant growth promoting substances with significant impact on root development and opens the possibility of using
these compounds for improved plant production.

Alkamides are secondary metabolites comprising
over 200 related compounds widely distributed in
plants. These amides have been found in as many as
10 plant families: Aristolochiaceae, Asteraceae, Bras-
sicaceae, Convolvulaceae, Euphorbiaceae, Menisper-
maceae, Piperaceae, Poaceae, Rutaceae, and So-
lanaceae. Several species containing high levels of
alkamides are found in the Asteraceae, Piperaceae,
and Rutaceae (Christensen and Lam, 1991; Kashiwada
et al., 1997; Parmar et al., 1997). The general structure
of alkamides originates from the condensation of an
unsaturated fatty acid and an amine (Hofer et al.,
1986). Although different chain length alkamides have
been found in plants, most of them contain a 2E dou-
ble bond conjugated to the amide group substituted
with a N-isobutyl group (Rios-Chavez et al., 2003).
Increasing interest has being paid to alkamides
because of their wide range of biological activities
(Jacobson, 1971). Many of the plant species contain-
ing these compounds have been used in traditional
medicine of different civilizations (Ximenez, 1615).
They are recognized for their pungent taste and for
causing numbing and salivation. Insecticidal proper-
1
This work was supported in part by the Howard Hughes
Medical Institute (grant no. Nbr55003677) and by the European
Commission (grant no. ICA– 4 –CT2000 –30017).
2
These authors contributed equally to this work.
* Corresponding author; e-mail lherrera@ira.cinvestav.mx; fax
462– 624 –58 – 46.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.103.034553.
1058 Plant Physiology, March 2004, Vol. 134, pp. 1058–1068, www.plantphysiol.org © 2004 American Society of Plant Biologists
ties have also been attributed to alkamides such as
affinin (Jacobson, 1971). However, it is completely
unknown whether alkamides could have a role in
plant growth and differentiation.
Amidenin, a nonsubstituted alkamide isolated
from the actinomycete fungus Amycolatopsis sp. was
reported to stimulate the growth of rice (Oryza sativa)
seedlings (Kanbe et al., 1993), although little infor-
mation is available about the mode of action of ami-
denin and no information is available about any po-
tential growth regulating activity of alkamides
produced by plants.
In the present work, the effects of three plant alka-
mides (N-isobutyl-2E,6Z,8E-decatrienamide, N-iso-
butyl-2E-decenamide, and N-isobutyl-decanamide)
on the growth and development of Arabidopsis seed-
lings are presented. We provide evidence that alka-
mides can act as plant growth-promoting substances.
Developmental alterations induced by alkamides in-
cluded greater formation and emergence of lateral
roots and increased root hair elongation. The in-
volvement of auxin in mediating the observed activ-
ity of alkamides was tested using the auxin respon-
sive marker genes DR5:uidA and BA3:uidA and
auxin-signaling Arabidopsis mutants.
RESULTS
Isolation and Purification of Alkamides
Affinin (N-isobutyl-2E,6Z,8E-decatrienamide), a
member of the alkamides, is present in Heliopsis lon-

gipes roots in concentrations as high as 1% (w/w) on
a fresh weight basis (Molina-Torres et al., 1996),
opening the possibility of using this compound to
investigate further the effects of alkamides on plant
growth and the physiological mechanisms by which
they act. The alkamides N-isobutyl-2E-decenamide
and N-isobutyl-decanamide were obtained by cata-
lytic reduction of affinin (See “Materials and Meth-
ods”). Figure 1 shows the structural features of the
molecules whose effects on plant growth and devel-
opment were analyzed in this work.
Effect of Affinin on Growth of Arabidopsis Seedlings
We tested the effect of affinin on the growth of
Arabidopsis (Colombia 0 [Col-0] ecotype), by grow-
ing plants on 0.1⫻ Murashige and Skoog solid me-
dium containing different concentrations of this com-
pound. After 12 d of growth, treatments of 7 ⫻ 10⫺6
and 1.4 ⫻ 10⫺5 m affinin stimulated shoot and root
biomass production (Fig. 2, A–C). Treatments of
2.8 ⫻ 10⫺5, 5.6 ⫻ 10⫺5, and 1.2 ⫻ 10⫺4 m affinin had
an inhibitory effect on Arabidopsis growth (Fig. 2,
Figure 1. Affinin and major catalytic reduction alkamide structures. A,
N-isobutyl-2E,6Z,8E-decatrienamide; B, N-isobutyl-2E-decenamide,
major intermediate at 3 min reduction time; and C, N-isobutyl-
decanamide, final reduction product.
Plant Physiol. Vol. 134, 2004
Alteration of Plant Development by Alkamides
D–F). In particular, affinin treatments had a dramatic
effect on the Arabidopsis root system architecture by
altering primary root growth and lateral root forma-
tion (Fig. 2, A–F).
To more closely analyze the effects of affinin on
plant development, primary root length, number of
emerged lateral roots, and lateral root density were
determined in Arabidopsis seedlings grown in 0.1⫻
Murashige and Skoog media supplemented with var-
ious concentrations of affinin. After 8 d of growth,
primary root growth promotion was observed at con-
centrations of 7 ⫻ 10⫺6 and 1.4 ⫻ 10⫺5 m affinin,
whereas primary root inhibition could be detected at
concentration of 5.6 ⫻ 10⫺5 m. At this affinin concen-
tration, the primary root length was 43% lower than
that registered for the controls (Fig. 3A). Affinin con-
centrations in the range of 7 ⫻ 10⫺6 to 5.6 ⫻ 10⫺5 m
increased by 2-fold the number of lateral roots (Fig.
3B). The density of lateral roots was also calculated
by dividing the number of lateral roots by the length
of the primary root to normalize for the effects of
affinin on primary root length. Lateral root density
dramatically increased with increased affinin concen-
tration in the media, being up to 3.5-fold higher in the
1.2 ⫻ 10⫺4 m affinin treatment when compared with
the untreated controls (Fig. 3C).
Effect of Affinin on Lateral Root Primordia Initiation
The effect of affinin concentrations increasing the
number of lateral roots could be due to a stimulation
of the emergence of pre-existing lateral root primor-
dia or to the de novo formation of additional root
primordia. To establish the developmental basis for
the effect of affinin on lateral root development, lat-
eral root primordia (LRP) originating in the Arabi-
dopsis primary root were quantified at d 6 after
germination. Seedling roots were first cleared to en-
able LRP at early stages of development to be visu-
alized and counted. Each LRP was classified accord-
ing to its stage of development as reported by Zhang
et al. (1999), who consider the following stages: stage
A, up to three cell layers; stage B, unemerged, of
more than three cell layers; stage C, LR emerged of
less than 0.5 mm in length; stage D, lateral root larger
than 0.5 mm.
The lateral root density analysis revealed that
plants treated with 7 ⫻ 10⫺6 and 1.4 ⫻ 10⫺5 m affinin,
respectively, formed 55% and 71% more LRP than the
control (Fig. 4A). The stage distribution of primordia
was also affected by affinin. Treatments of 7 ⫻ 10⫺6
and 1.4 ⫻ 10⫺5 m affinin increased the number of LRP
in stages B through D compared with the control
(Fig. 4B). An even greater interval of affinin treat-
ments (7 ⫻ 10⫺6–5.6 ⫻ 10⫺5 m) increased the number
of LRP that had emerged from the primary root
(stage D; Fig. 4B). The observations that affinin treat-
ments increased primordia density and the transition
of LRP from stage A to further stages of development
1059
Ramı́rez-Chávez et al.
Figure 2. Effects of affinin on Arabidopsis growth. Wild-type Col-0 seedlings were grown for 10 d under increasing affinin
concentrations on vertically oriented agar plates. Control (A), 7 ⫻ 10⫺6 (B), 1.4 ⫻ 10⫺5 (C), 2.8 ⫻ 10⫺5 (D), 5.6 ⫻ 10⫺5 (E),
and 1.2 ⫻ 10⫺4 M (F) affinin. All photographs are at the same magnification. Scale bar ⫽ 1 cm.
suggest that affinin can promote root branching by
inducing lateral root primordia initiation and pro-
moting the emergence of lateral roots.
Effect of Alkamides on Root Hair Development
Root hairs are root epidermal cells that increase the
total absorptive surface of the root system and par-
ticipate in nutrient and water uptake. To analyze if
alkamides could alter root hair development, we per-
formed experiments in which Arabidopsis plants
were grown on the surface of agar plates with 0.1⫻
Murashige and Skoog medium and different concen-
trations of alkamides.
Root hair parameters were analyzed at d 6 after
germination on primary roots of control and
alkamide-treated plants. Affinin treatments substan-
tially increased root hair length (Fig. 5, A and B). A
significant difference in root hair length could be
observed at treatment of 7 ⫻ 10⫺6 m affinin. How-
ever, the most important effect was observed in roots
grown under 5.6 ⫻ 10⫺5 m affinin. Under our exper-
imental conditions, root hairs of control plants were
approximately 0.1 mm long, whereas those of plants
treated with 5.6 ⫻ 10⫺5 m affinin were approximately
0.5 mm in length, about five times the length of
control root hairs.
The effect of N-isobutyl-2E-decenamide and
N-isobutyl-decanamide on root hair growth was also
tested. Both molecules, obtained by chemical reduc-
tion of affinin, stimulated root hair elongation at
concentrations between 10⫺8 to 10⫺5 m (Fig. 6, A–T).
Concentrations higher than 10⫺8 m N-isobutyl-2E-
decenamide stimulated root hair elongation close to
the root tip (Fig. 6, A–E). The stimulatory effect of
1060
this compound on root hair growth was most clearly
observed when mature regions of the primary root
were compared (Fig. 6, F–J). Similar effects were
observed with treatments of N-isobutyl-decanamide
(Fig. 6, K–T). Treatments of 1 ⫻ 10⫺7 m N-isobutyl-
2E-decenamide and N-isobutyl-decanamide had a 4-
to 5-fold increase in hair length when compared with
the control (Fig. 6, H and R). These results indicate
that several alkamides can regulate root hair growth,
some of which have greater activity than affinin on
root hair elongation.
Effects of Affinin on Cell Division and Elongation
To test whether Alkamide treatments could alter
root development by altering cell division and/or
elongation, we analyzed the expression of the cell
cycle marker CycB1:uidA (Colón-Carmona et al.,
1999) in transgenic plants growing under different
affinin treatments. The location and intensity of
CycB1:uidA expression in the 7 ⫻ 10⫺6 M affinin
treatment was similar to that observed in untreated
seedlings, showing typical patchy expression in the
meristem (Fig. 7, A and B). Interestingly, in the treat-
ments of 5.6 ⫻ 10⫺5 and 1.2 ⫻ 10⫺4 M affinin the
number of cells showing CycB1:uidA expression was
clearly reduced (Fig. 7, C and D). We also analyzed
whether cell elongation was altered by alkamides by
measuring the epidermal cell size in the differentia-
tion and maturation regions of primary roots in WT
(Col-0) plants. These measurements showed that epi-
dermal cells in plants treated with 5.6 ⫻ 10⫺5 and
1.2 ⫻ 10⫺4 M affinin are in average 49% and 63%
shorter than those of wild type (Fig. 7, E–G). There-
fore, the observed inhibitory effect of affinine on
Plant Physiol. Vol. 134, 2004

Figure 3. Effects of affinin on Arabidopsis root architecture. Wild-
type Col-0 seedlings were grown for 10 d under increasing affinin
concentrations on vertically oriented agar plates. Data are given for
the length of the primary root (A), lateral root number (B), and lateral
root density (C). Values shown represent the mean of 20 seedlings ⫾
SD. Different letters represent means statistically different at the 0.05
level.
primary root elongation is caused both by a reduc-
tion in the cell proliferating activity in the meristem
and by an inhibition of cell elongation.
Effect of Affinin on Auxin-Inducible Gene Expression
The observed effect of affinin on several aspects of
root development is similar to that described for
auxin in most plant species, including Arabidopsis.
Auxins such as indole acetic acid enhance primary
root growth at low concentrations, and at high con-
Plant Physiol. Vol. 134, 2004
Alteration of Plant Development by Alkamides
centrations, promote root hair and lateral root forma-
tion and elongation but inhibit primary root growth
(Blakely et al., 1982; Laskowski et al., 1995). To test
whether affinin could alter auxin-regulated gene ex-
pression in roots, and in this way affect the root
system architecture, we conducted analyses of the
expression of the ␤-glucuronidase (GUS) reporter
gene in Arabidopsis lines harboring the DR5:uidA
and BA3:uidA gene constructs. Both reporter lines
have proved to be useful in studying auxin-regulated
gene expression in Arabidopsis (Ulmasov et al., 1997;
Oono et al., 1998). Figure 8 shows histochemical
staining for transgenic DR5:uidA and BA3:uidA
plants that were grown 7 d under auxin (2,4-
dichlorophenoxyacetic acid [2,4-D]) or affinin treat-
ments. As previously reported (Ulmasov et al., 1997),
in untreated control plants, DR5:uidA is expressed
primarily in the columella and quiescent center (Fig.
Figure 4. Effects of affinin on Arabidopsis lateral root development.
A, Density of lateral roots; B, lateral root stage distribution. Six-day-
old primary roots were cleared and the number and stage of LRP
were recorded. The results show the number of primordia at each
developmental stage according to Zhang et al. (1999), for 10 indi-
vidual roots. This analysis was repeated twice with similar results.
1061
Ramı́rez-Chávez et al.
Figure 5. Effect of affinin on root hair formation. A, Average root hair
length of fully expanded root hairs. B, Light microscope images of
control and affinin induced-root hairs (magnification ⫽ 60⫻).
8A). DR5:uidA plants grown under a concentration of
10⫺8 m 2,4-D showed GUS activity throughout the
primary root (Fig. 8B). A similar pattern of expres-
sion has been previously reported (Ulmasov et al.,
1997; Sabatini et al., 1999). In contrast, the pattern of
GUS expression in DR5:uidA seedlings treated with
up to 1.2 ⫻ 10⫺4 m affinin remained similar to that
observed in untreated controls (Fig. 8, C and D).
Untreated BA3:uidA plants did not show detectable
levels of GUS activity (Fig. 8E), whereas when treated
with 10⫺8 m 2,4-D, they showed GUS expression
mainly in the root elongation zone (Fig. 8F), similarly
to that reported by Oono et al., (1998). In contrast,
BA3:uidA seedlings failed to show GUS staining in
the root elongation zone even at the highest level of
affinin tested (10⫺4 m; Fig. 8, G and H). These results
show that affinin treatments can induce root growth
alterations without altering the expression of auxin-
inducible genes.
Alkamide Response in Auxin-Signaling Mutants
The results from DR5:uidA and BA3:uidA marker
expression analyses indicated that affinin effects
could not be related to auxin-altered responsive gene
1062
expression. To date, different auxin-resistant mutants
have been isolated, which represent a valuable tool in
investigating at the genetic level any potential role of
the auxin-signaling pathway in mediating the root
developmental changes induced by alkamides. With
this in mind, the effect of 5.6 ⫻ 10⫺5 m affinin on
primary root growth of the aux1-7, eir1-1, and axr4-2
auxin mutants was tested. As shown in Figure 9,
treatment with 5.6 ⫻ 10⫺5 m affinin caused 40%
inhibition in primary root growth in WT plants of the
Col-0 and Wassilewskija ecotypes compared with un-
treated control plants. Under our experimental con-
ditions, in media lacking affinin, the three auxin mu-
tants showed longer primary roots compared with
WT plants. Despite this difference in primary root
growth, when aux1-7, eir1-1, and axr4-2 plants were
grown in the presence of 5.6 ⫻ 10⫺5 m affinin, an
inhibition in root elongation was observed (Fig. 9).
Because root elongation in auxin-resistant mutants
was inhibited to a similar extent than the WT, we
conclude that these mutants are equally sensitive to
high affinin concentrations than WT plants.
DISCUSSION
Effects of Alkamides on Plant
Growth and Development
Results from recent research suggest the existence
of a wide range of molecules of diverse origin that
could alter plant development, and some of these
include compounds with fatty acid moieties such as
sphingolipids and alkamides (Worral et al., 2003).
Kanbe et al. (1993) showed that amidenin, a nonsub-
stituted alkamide isolated from an actinomycete fun-
gus, could stimulate growth of rice seedlings at con-
centrations from 0.6 ⫻ 10⫺5 to 1.8 ⫻ 10⫺5 m.
However, no information is available about the spe-
cific effects of amidenin, and, to the best of our
knowledge, no alkamide of plant origin has been
tested for plant growth regulating activity. Here, we
report that affinin, a plant alkamide alters the growth
and development of Arabidopsis seedlings. Affinin
treatments in the 7 ⫻ 10⫺6 to 2.8 ⫻ 10⫺5 m range,
enhanced early growth of Arabidopsis, stimulated
lateral root formation, and promoted root hair elon-
gation. Analyses of LRP in cleared primary roots
showed that affinin treatments can induce the forma-
tion of lateral roots by inducing perycicle cells to
divide and form new LRP, and by stimulating the
emergence of existing LRP. The effects of affinin in
stimulating biomass production and lateral root de-
velopment have also been observed in tobacco (Nico-
tiana tabacum) and rice plants (data not shown), sug-
gesting that some alkamides could have general
plant growth regulating activity.
Treatments with 5.6 ⫻ 10⫺5 to 1.2 ⫻ 10⫺4 m affinin
inhibited primary root growth and 1.2 ⫻ 10⫺4 m
affinin also inhibited root hair elongation. These
treatments were found to alter cell division and elon-
Plant Physiol. Vol. 134, 2004
 Alteration of Plant Development by Alkamides

Figure 6. Effects of N-isobutyl-2E-decenamide and N-isobutyl-decanamide on epidermal cell differentiation. The root hairs
forming at the primary root tip region (A–E and K–O) and in mature region (F–J and P–T) of the root of 6-d-old Arabidopsis
seedlings were photographed. Photographs are representative individuals of at least 40 plants analyzed. Scale bar ⫽ 500 ␮m.

Plant Physiol. Vol. 134, 2004 1063

Ramı́rez-Chávez et al.

Figure 7. Effects of affinin on cell proliferating activity and cell elongation. Wild-type Col-0 and CycB1:uidA seedlings were
grown for 7 d under varied affinin concentrations on vertically oriented agar plates, were stained for GUS activity, and were
cleared to measure cell size. A, CycB1:uidA primary root meristem of a plant grown in medium without affinin; B, 7 ⫻
10⫺6 M affinin; C, 5.6 ⫻ 10⫺5 M affinin; D, 1.2 ⫻ 10⫺4 M affinin. E, Mean trichoblast cell length in Col-0 plants treated with
or without 5.6 ⫻ 10⫺5 and 1.2 ⫻ 10⫺4 M affinin. F, Light microscope images of control and 1.2 ⫻ 10⫺4 M affinin
treated-epidermal cells. CycB1:uidA photographs are representative individuals of at least 20 plants stained. Scale bar in A
through D ⫽ 100 ␮m and in F and G ⫽ 50 ␮m.

gation in Arabidopsis primary roots (Fig. 7, A–G).
Because the most important stimulating effect of af-
finin on root hair elongation occurred at a concentra-
tion that inhibits primary root growth (5.6 ⫻ 10⫺5 m),
the possibility is raised that the observed enhanced
root hair elongation might be due to the formation of
shorter epidermal cells. However, our data shows
that root hair development is significantly stimulated
at low affinin concentrations (7 ⫻ 10⫺6 m) in which
primary root growth is stimulated. In addition, we
observed that treatment with 1.2 ⫻ 10⫺4 m affinin
that showed the greatest inhibitory effect on epider-
mal cell elongation also inhibited root hair growth
when compared with lower affinin treatments (Figs.
5B and 7E). Therefore, the effects of affinin on root
hair growth cannot be just explained as an epidermal
cell growth compensation effect. Therefore, we pro-
pose that the effect of affinin on root hair elongation
is, at least in part, mediated by a specific signaling
mechanism yet to be identified.
1064
The alkamides N-isobutyl-2E-decenamide and
N-isobutyl-decanamide are naturally present in Ac-
mella radicans and Cissampelos glaberrima (Laurerio-
Rosario et al., 1996; Rios-Chavez et al., 2003). How-
ever, to have enough material for our assays, these
amides were obtained by chemical reduction of affi-
nin. The most important difference in the structure of
these molecules, compared with affinin, is the ab-
sence of the 6, 8-conjugated double bonds in both,
and the absence of a 2E-conjugated double bond in the
latter (Fig. 1). These compounds were found to stim-
ulate root hair growth at a concentration of 10⫺8 m, a
concentration 100 to 1,000 times lower than that re-
quired of affinin to stimulate root hair growth (Fig. 6).
Alkamides Alter Root Development by an Auxin-
Independent Mechanism
The phytohormone auxin is a key regulator of lat-
eral root development and root hair formation
Plant Physiol. Vol. 134, 2004
 Alteration of Plant Development by Alkamides

Figure 8. Effect of affinin on auxin-regulated gene expression. A, Twelve hours of GUS staining of DR5:uidA primary roots
grown for 7 d in medium without auxin; B, under 10⫺8 M 2,4-D; C, treatments of 2.8 ⫻ 10⫺5 M affinin; C, 1.2 ⫻ 10⫺4 M
affinin. E, Twelve hours of GUS staining of BA3:uidA primary roots grown for 7 d in medium without auxin, under 10⫺8 M
2,4-D (F), and treatments of 2.8 ⫻ 10⫺5 M affinin (G) and 1.2 ⫻ 10⫺4 M affinin (H). Photographs are representative individuals
of at least 20 plants stained. Scale bar ⫽ 200 ␮m.

(Blakely et al., 1982; Laskowski et al., 1995). Auxin is
synthesized in the shoot system and is transported to
the root through the action of specific polar auxin
transport systems. Recently, it has been demon-
strated that basipetal and acropetal auxin transport
are required during the initiation and emergence
Figure 9. Primary root growth response of wild-type (Wassilewskija
and Col-0) and auxin-resistant mutants to affinin. Seedlings were
grown for 12 d on nutrient media with or without 5.6 ⫻ 105 M affinin
on vertically oriented agar plates. Values shown represent the mean
primary root length of 20 seedlings ⫾ SD. Different letters are used to
indicate means that differ significantly at the 0.05 level. The exper-
iment was repeated twice with similar results.
Plant Physiol. Vol. 134, 2004
phases of lateral root development (Casimiro et al.,
2001; Bhalerao et al., 2002). Auxins are active over a
very wide range of concentrations: low auxin concen-
trations (10⫺10–10⫺9 m) stimulate primary root
growth, whereas higher concentrations (10⫺8–10⫺6
m) inhibit primary root growth and stimulate lateral
root and root hair formation. An important differ-
ence in the mode of action of alkamides compared
with auxins is that whereas auxins induce lateral root
formation at concentrations that have an inhibitory
effect on primary root growth (10⫺8–10⫺7 m range),
alkamides can induce lateral root formation at a con-
centration that enhances primary root growth (10⫺6–
10⫺5 m). These observations suggest that alkamides
could alter root growth by a different mechanism to
that of auxins.
Further support of the hypothesis that alkamides
act by an auxin-independent signaling pathway came
from two different lines of evidence: expression stud-
ies of the auxin-inducible markers DR5:uidA and
BA3:uidA, and the primary root growth inhibition of
the auxin mutants aux1-7, eir1-1, and axr4-2 when
grown under a high affinin concentration. In our
experiments, treatment with 10⫺8 m 2,4-D induced
DR5:uidA and BA3:uidA expression, whereas concen-
trations of up to 10⫺4 m affinin failed to induce these
auxin-inducible gene markers (Fig. 8). N-isobutyl-2E-
decenamide and N-isobutyl-decanamide also failed
to induce auxin-regulated expression of DR5:uidA
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Ramı́rez-Chávez et al.
and BA3:uidA (data not shown). These results, to-
gether with the finding that the primary root growth
of the auxin-resistant mutants aux1-7, eir1-1, and
axr4-2 is equally sensitive to affinin than the WT (Fig.
9), indicate that growth effects and developmental
changes induced by alkamides are not mediated by
the known auxin-signaling pathways.
Alkamides: New Players in Plant Signaling?
Although no data is available about the site of
biosynthesis or transport mechanisms of alkamides
in plants, taking our data together with those from
the literature, it is tempting to speculate that, like
endogenous auxins (Abel et al., 1994), alkamides may
affect particular signal transduction cascades that
modify root growth and differentiation. Because the
effect of affinin in stimulating root hair elongation is
similar to that reported for ethylene (Tanimoto et al.,
1995), one possibility is that alkamides could alter
ethylene biosynthesis/perception in some way.
However, in contrast to ethylene that induces the
ectopic formation of root hairs (Tanimoto et al., 1995;
Cao et al., 1999), alkamides apparently only induce
root hair elongation in trichoblast cells. Further ex-
perimentation to determine whether alkamides influ-
ence ethylene production or alter its signaling path-
way is required.
Very recently, the sphingolipids, a group of mole-
cules that include ceramide (Fig. 10) and
sphingosine-1-phosphate with well-known regula-
tory functions in animal and fungal species, have
been found to play an important role in plant devel-
opment (Worral et al., 2003). For example, it has been
found that drought and abscisic acid can affect the
aperture of the stomatal pore by the action of
sphingosine-1-phosphate (Ng and Hetherington,
2001). Based on their chemical structure, the alka-
mides studied in this work are somewhat structurally
related to the sphingolipids ceramide and glucosyl-
ceramide (Figs. 1 and 10). In affinin, the decamide
resembles the acidic moiety of ceramide, albeit hav-
ing a shorter C10 instead of the C18 chain in cer-
amide (Fig. 10). Whether alkamides and sphingolip-
Figure 10. Structures of ceramide and N-isobutyl-decanamide.
1066
ids have similar biological activities or act through
common signaling pathways remains to be deter-
mined. The possibility that a breakdown product of
ceramide or other sphingolipids may result in metab-
olites with the observed function of alkamides cannot
be excluded.
The data presented in this work and other recent
evidence from the literature suggest the existence of
new plant growth-regulating substances yet to be
characterized that may include alkamides and sphin-
golipids. To our knowledge, this is the first report of
plant-produced alkamides as regulators of root de-
velopment. Whether these compounds can alter other
aspects of plant development would be an interesting
field for future research.
MATERIALS AND METHODS
Plant Material and Growth Conditions
Heliopsis longipes (Gray) Blake (Asteraceae) plants were collected at an
altitude of 2,500 m above sea level in the municipality of Xichú, located in
the Sierra Gorda of Guanajuato State, central México. Voucher specimens
(H. longipes Jorge Molina Torres, Instituto de Ecologı́a del Bajı́o) were
deposited at the Instituto de Ecologı́a (Pátzcuaro, México).
Arabidopsis ecotype Col-0 and the Arabidopsis transgenic lines DR5:
uidA (Ulmasov et al., 1997), BA3:uidA (Oono et al., 1998), and CycB1:uidA
(Colón-Carmona et al., 1999) were used for all experiments unless indicated
otherwise. Arabidopsis mutant eir1 (Roman et al., 1995) was kindly pro-
vided by Dr. Plinio Guzmán (Departamento de Ingenierı́a Genética, Centro
de Investigación y Estudios Avanzados del Instituto Politécnico Nacional,
Irapuato, Gto. México). aux1-7 (Pickett et al., 1990) and axr4-2 (Hobbie and
Estelle, 1995) were kindly provided by Dr. Claire Grierson (School of Bio-
logical Sciences, University of Bristol, Bristol, UK). Seeds were surface
sterilized with 95% (v/v) ethanol for 5 min and with 20% (v/v) bleach for
7 min. After five washes in distilled water, seeds were germinated and
grown on agar plates containing 0.1⫻ Murashige and Skoog medium, pH
5.7, 0.5% (w/v) Suc, and 1% (w/v) agar (López-Bucio et al., 2002). The basic
medium contained 2.0 mm NH4NO3, 1.9 mm KNO3, 0.3 mm CaCl2.2H2O,
0.15 mm MgSO4.7H2O, 5 ␮m KI, 25 ␮m H3BO3, 0.1 mm MnSO4.H2O, 0.3 mm
ZnSO4.7H2O, 1 ␮m Na2Mo04.2H2O, 0.1 ␮m CuSO4.5H2O, 0.1 ␮m
CoCl2.6H2O, 0.1 mm FeSO4.7H2O, 0.1 mm Na2EDTA.2H2O, inositol (10 mg
L⫺1), and Gly (0.2 mg L⫺1).
Plates were placed vertically at an angle of 65° to allow root growth along
the agar surface and to allow unimpeded growth of the hypocotyl into the
air. For plant growth, we used a plant growth cabinet (Percival Scientific,
Perry, IA) with a photoperiod of 16 h of light, 8 h of darkness, a light
intensity of 300 ␮mol m⫺2 s⫺1, and temperature of 22°C to 24°C.
Isolation of Affinin
H. longipes roots (1 kg) were ground and extracted in 10 L of ethanol at
room temperature for 1 week. The alcoholic extract was evaporated to
dryness, dissolved in hexane, and fractionated in a 50-cm open glass column
packed with silica gel mesh 200 (J.T. Baker, Paris, KY). Different fractions
were eluted with 100 mL of hexane and increasing polarity mixtures of
hexane:ethyl acetate: 200 mL (90:10, v/v), 200 mL (80:20), 100 mL (70:30), 100
mL (60:40), and 100 mL (50:50), and 100 mL of ethyl acetate. Fifty-milliliter
fractions were collected, evaporated, and dissolved in 1 mL of ethanol and
were analyzed by capillary gas chromatography (GC) coupled to a mass
selective (MS) detector. Fractions eluted with hexane:ethyl acetate (70:30)
contained affinin that was further quantified as reported previously
(Molina-Torres et al., 1996). Affinin was purified by column chromatogra-
phy and three times by thin-layer chromatography (TLC) to obtain only one
main peak on GC/MS (data not shown). At this stage of purification, affinin
comprised 99.6% (w/v) of the total extract, including two affinin stereoiso-
mers (produced by trans-isomerization of one or the two double bonds
present in affinin), and methyl-affinin (N-2 methylbutyl 2E,6Z,8E decatrien-
Plant Physiol. Vol. 134, 2004

amide) comprising the remaining 0.4% (w/v) of the extract. No other com-
pounds could be detected in the affinin extract by GC-MS analysis. For the
reduced amides, N-isobutyl-2E-decenamide and N-isobutyl-decanamide, pu-
rity was 100% because affinin and its stereoisomers were reduced to the same
component and were totally separated from methyl-affinin by argented TLC.
GC/MS Analysis
Samples were analyzed in a gas chromatograph (model 5890; Hewlett-
Packard, Palo Alto, CA) equipped with a capillary column HP-1MS (30- ⫻
0.25-mm i.d; 0.25-␮m film thickness) coupled to a MS detector (model 5972;
Hewlett-Packard). Operating conditions were an injector temperature of
200°C, and the oven temperature was programmed with an initial temper-
ature of 150°C for 3 min, increasing at the rate of 4°C min⫺1 to a final
temperature of 300°C, which was maintained for 20 min. Helium was used
as carrier gas with a constant flow of 1 mL min⫺1. One microliter of the
sample was injected with a split ratio of 50:1.
Production of N-Isobutyl-2E-Decenamide and
N-Isobutyl-Decanamide
To obtain the N-isobutyl-2E-decenamide and N-isobutyl-decanamide, a
catalytic reduction of affinin was performed. Ten milligrams of platinum
oxide were added to 10 mg of affinin resuspended in 2 mL of ethanol. A
hydrogen gas stream was bubbled through the mixture maintained at 80°C.
After 3 min of reduction, the 2E-decanamide was collected as major com-
ponent and was purified by analytical argented TLC 20- ⫻ 20-cm glass
plates as reported elsewhere (Rios-Chavez et al., 2003). Plates were developed
using a hexane:ethyl acetate (2:1, v/v) as a solvent system. Developed plates
were solvent freed at room temperature and were then sprayed with a 0.02%
(w/v) ethanolic fluorescein solution. The N-isobutyl-2E-decenamide was de-
tected as a fluorescent band on a dark background with a relative retention
index of 0.7. This band was collected from the plate and was eluted with ethyl
acetate that was later evaporated under a stream of nitrogen. The TLC
purification was repeated three times and the fraction containing the inter-
mediary was submitted to GC/MS detection, giving only one peak. The
N-isobutyl-decanamide was obtained after a 7-min reduction as the only
product. The platinum oxide was centrifuged, and the supernatant was
decanted, evaporated to dryness, dissolved in hexane, and submitted to
GC/MS detection, giving only one peak. Compounds were initially identified
as N-isobutyl-2E-decenamide and N-isobutyl-decanamide by their mass spec-
tra. 1H NMR and 13C NMR analysis on a model Gemini 200 (Varian, Palo
Alto, CA) further confirmed the reported structures.
Histological Analysis
For histochemical analysis of GUS activity, Arabidopsis seedlings were
incubated overnight at 37°C in a GUS reaction buffer (0.5 mg mL⫺1
5-bromo-4-chloro-3-indolyl-B-d-glucuronide in 100 mm sodium phosphate,
pH 7). The stained seedlings were cleared by the method of Malamy and
Benfey (1997). For each marker line and for each treatment, at least 10
transgenic plants were analyzed. A representative plant was chosen for each
affinin or auxin treatment and was photographed using the Nomarski optics
on a microscope (DMR; Leica, Deerfield, IL).
Data Analysis
Arabidopsis root systems were viewed with a stereomicroscope (AFX-
II-A; Nikon, Tokyo). All lateral roots emerged from the primary one, were
observed at the 3⫻ objective, and were taken into account for lateral root
number data. Primary root length was determined for each root using a
plastic ruler. The stages of lateral root primordia were analyzed on cleared
roots using a microscope (UW; Nikon) in the 40⫻ objective. For trichoblast
measurements, cleared root images from the mature zone were recorded
with a digital camera connected with a microscope. From the same zone, the
lengths of 10 mature epidermal cells per root were photographed and the
cell length was then measured with Image-Pro Plus version 4.0 for Windows
(Image-Pro Plus; Media Cybernetics, Silver Spring, MD). The measurements
were repeated in at least eight plants from two independent experiments.
Plant Physiol. Vol. 134, 2004
Alteration of Plant Development by Alkamides
For all experiments, the overall data was statistically analyzed with the
SPSS 10 program (SPSS Inc., Chicago). Univariate and multivariate analyses
with a Tukey’s post hoc test were used for testing differences in primary
root length, lateral root number, and lateral root density under affinin
treatments in WT and mutant plants. Different letters are used to indicate
means that differ significantly (P ⬍ 0.05).
ACKNOWLEDGMENTS
We thank Cristina Reynaga-Peña for her support in microscopy. We also
thank Claire Grierson, Athanasios Teologis, Tom Guilfoyle, Peter Doerner,
and Plinio Guzmán for kindly providing us with seeds of transgenic and
mutant lines.
Received October 7, 2003; returned for revision October 29, 2003; accepted
December 9, 2003.
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