REVIEW: PART OF A SPECIAL ISSUE ON PALM BIOLOGY

Environmental regulation of sex determination in oil palm: current knowledge

and insights from other species
Hele`ne Adam
1
, Myriam Collin
1
, Frederique Richaud
2
, Thierry Beule
2
, David Cros
3
, Alphonse Omore
4
,
Lei Nodichao
4
, Bruno Nouy
5
and James W. Tregear
1,
*
1
IRD,
2
CIRAD, IRD/CIRAD Palm Developmental Biology Group, UMR DIADE, Centre IRD, 911 avenue Agropolis, 34394
Montpellier, France,
3
CIRAD, UMR AGAP,
4
INRAB, Station INRAB, BP1, Pobe`, Benin and
5
PalmElit SAS, Parc Agropolis Bt.
14, 2214 Boulevard de la Lironde, 34980 Montferrier sur Lez, France
* For correspondence. E-mail james.tregear@ird.fr
Received: 16 February 2011 Returned for revision: 9 March 2011 Accepted: 7 April 2011 Published electronically: 28 June 2011
Background The African oil palm (Elaeis guineensis) is a monoecious species of the palm subfamily
Arecoideae. It may be qualied as temporally dioecious in that it produces functionally unisexual male and
female inorescences in an alternating cycle on the same plant, resulting in an allogamous mode of reproduction.
The sex ratio of an oil palm stand is inuenced by both genetic and environmental factors. In particular, the
enhancement of male inorescence production in response to water stress has been well documented.
Scope This paper presents a review of our current understanding of the sex determination process in oil palm
and discusses possible insights that can be gained from other species. Although some informative phenological
studies have been carried out, nothing is as yet known about the genetic basis of sex determination in oil palm,
nor the mechanisms by which this process is regulated. Nevertheless new genomics-based techniques, when com-
bined with eld studies and biochemical and molecular cytological-based approaches, should provide a new
understanding of the complex processes governing oil palm sex determination in the foreseeable future.
Current hypotheses and strategies for future research are discussed.
Key words: Sex determination, oil palm, Elaeis guineensis, inorescence, monoecious, temporal dioecy,
owering.
INTRODUCTION
The physical separation of fertile male and female reproduc-
tive organs on different owers of the same plant or different
individuals of the same species is a condition known as
dicliny. Diclinous species are estimated to represent approx.
10 % of owering plants (Ainsworth, 2000). It is generally
considered that dicliny confers two main advantages: (1)
better use of available resources through the specialization of
the pollen and ovule producing reproductive organs, and (2)
the enhancement of heterosis (hybrid vigour) through the
promotion of outcrossing (allogamy). Dicliny can be divided
into two main categories: monoecy, in which male (staminate)
and female ( pistillate) owers are produced on the same plant;
and dioecy, in which single-sex male and female plants exist.
Other more complex situations involving the co-occurrence of
male, female and bisexual owers or plants are collectively
classied as polygamy.
Although spatial separation of male and female reproductive
organs is employed by many species as a means to achieve
outbreeding, a similar result can be achieved by temporal
separation. This strategy, referred to by Cruden and
Hermann-Parker (1977) as temporal dioecism, was dened
as: in essence, a owering sequence which is synchronized
so there is little or no overlap between staminate and pistillate
phases of an individual plant. In its simplest form, temporal
dioecy can be achieved simply by the delayed maturation of
either carpels (in the case of protandrous owers) or stamens
(in the case of protogynous owers), two variants of the con-
dition known as dichogamy. It is interesting to note that many
self-incompatible plant species display dichogamous oral
development characters, implying that sexual interference
factors such as gamete wastage play a part in determining
tness and not just the promotion of outbreeding (Barrett,
2002).
The palm family (Arecaceae) is a particularly interesting
group in which to investigate dicliny and dichogamy, as both
are prevalent within this clade (Loo et al., 2006; Weiblen
et al., 2000). As regards spatial separation of the sexes, the
most common condition in palms is monoecy, which accounts
for approx. 65 % of genera in the family (Dranseld et al.,
2008) and which dominates in the Arecoideae, the largest
palm subfamily. The majority of monoecious palms produce
bisexual inorescences, with only a handful of exceptions,
notably in the genera Lepidorrachis, Arenga, Wallichia,
Wettinia, Marojejya, Attalea and Elaeis (Voeks, 1988; Baker
and Hutton, 2006).
The African oil palm (Elaeis guineensis) is the best studied
example of temporal dioecy in the palm family, due in particu-
lar to its economic importance as the worlds largest source of
vegetable oil. Considerable efforts have been devoted over the
last half century or so to improving the yield potential of cul-
tivated palms and to perfecting the growing conditions necess-
ary to realize this potential. Past studies have revealed that the
# The Author 2011. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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Annals of Botany 108: 15291537, 2011
doi:10.1093/aob/mcr151, available online at www.aob.oxfordjournals.org

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sex ratio of an oil palm stand is affected by both environ-
mental and genetic factors. In this paper, we rst discuss
current knowledge of the mode of action of environmental
factors on oil palm inorescence development, and then
discuss how they might act upon the plants reproductive
development to regulate sex determination.
I NFLORESCENCE DEVELOPMENT IN OI L PALM
As mentioned earlier, the oil palm normally produces func-
tionally unisexual male and female inorescences in an alter-
nating cycle, thereby minimizing the chances of
self-pollination. Macroscopic views of the normal male and
female inorescences are shown in Fig. 1. Male and female
inorescences are branched and easily distinguishable from
their overall structure, the male inorescence bearing a
larger number of rachillae (branches) inserted into the rachis
(central axis). Another easily recognizable difference
between the two sexes is in the form of the rachillae them-
selves; in the case of the female inorescence they bear a
distal sterile region that develops into a characteristic spine
not seen in the male rachilla. The early differentiation of oil
palm inorescences was studied by scanning electron
microscopy (SEM) by Van Heel et al. (1987) and in our
group using SEM and light microscopy on stained tissue sec-
tions (Adam et al., 2005). The earliest clear anatomical differ-
ence between the two sexes is the greater number of early
bracts arising at the base of spikelet primordia in the male
inorescence compared with its female equivalent (Van Heel
et al., 1987). This reects the larger number of owers
borne on the male rachilla and conrms the earlier conclusions
of Corley (1976a). A later key difference between the male and
female inorescences concerns the arrangement of owers on
the rachillae, the staminate owers being borne singly whereas
pistillate owers are anked by a pair of abortive accompany-
ing staminate owers. The female inorescence thus produces
oral triads, a structural arrangement that is dominant
(although not universal) in the subfamily Arecoideae. The
accompanying staminate owers develop pollen sacs with pro-
liferating microsporocytic cells; however, the latter display
incomplete development and the ower is shed soon after,
before the pistillate ower of the triad has reached maturity
(Adam et al., 2005). Both types of inorescence are protected
during their development by the prophyll and rst penduncular
bract, which are ruptured just before ower maturity is
reached. Other peduncular bracts are small, triangular struc-
tures which do not envelope the developing inorescence
(Dranseld et al., 2008).
I NFLORESCENCE DEVELOPMENT I N
RELATI ON TO VEGETATIVE GROWTH
In favourable growing conditions, an inorescence is initiated
in the axil of each leaf of the palm. The rate of leaf emission
varies with age, with typically three leaves produced per
month in young palms and two per month in the case of
older individuals. The development of an oil palm inores-
cence between the stages of initiation and ower maturity
lasts 23 years for both sexes, the process being initiated
soon after seedling establishment.
In tropical humid climates with regular rainfall, inores-
cence and fruit production is spread evenly throughout the
year. From an experimental point of view, this allows the
possibility of dissecting mature palms so as to visualize a com-
plete series of inorescence developmental stages within the
same individual. As a means of cross-referencing between
palms, inorescences are usually numbered according to
their subtending leaves. Thus, the youngest expanding leaf
with separating leaets (and its associated inorescence) is
designated as +1, the immediately older leaves being +2,
+3, etc., and younger leaves 0, 1, etc. A summary of the
key stages of inorescence development in relation to leaf
number is shown in Fig. 2.
I NSI GHTS I NTO THE CHRONOLOGY OF SEX
DETERMI NATI ON FROM FI ELD
OBSERVATI ONS
Typically, a mature palm will alternate between male and
female inorescence production during its lifetime; however,
the proportion of time spent in each phase will vary consider-
ably depending on both environmental and genetic factors.
The sex ratio is normally dened as the ratio of female to
total inorescences in a given group of palms. In regions
with high and regular rainfall (e.g. Malaysia and Indonesia),
oil palm sex ratios tend to vary little throughout the year, in
contrast to areas experiencing a marked dry season such as
in West Africa, where the sex ratio undergoes extensive uctu-
ations. Corley (1976a) noted that in the latter case, the period
of lowest sex ratio (high male inorescence production) occurs
during the rainy season and speculated that this character is an
adaptation against the reduction in airborne pollen density
caused by high atmospheric humidity. This illustrates the well-
established observation that oil palm sex determination is
strongly inuenced by climatic factors, with male inores-
cence production being promoted by water decit.
Male inorescence production can also be induced arti-
cially by defoliating adult plants, a technique which has
A B
FI G. 1. Macroscopic views of oil palm inorescences: (A) mature female
inorescence, (B) mature male inorescence. Scale bars 10 cm.
Adam et al. Environmental regulation of sex determination in oil palm 1530

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proved invaluable for increasing pollen production from pisi-
fera palms (Durand-Gasselin et al., 1999). The latter are
used as the male parent in most breeding programmes as
they are homozygous for the Sh thin shell thickness allele
and give rise to high oil-yielding tenera palms (Sh/Sh + )
when crossed with a thick-shelled dura (Sh + Sh + ) female
parent. It is interesting to note that pisifera palms produce
abortive fruit, which place lower demands on the plants
resources. As a result, pisifera palms usually remain female
when grown in conditions of high and regular water avail-
ability, thus creating the problem of limited pollen production
for breeding, which can be overcome by defoliation as men-
tioned earlier. The exact nature of the stress response under-
lying the induction of male inorescence production by
defoliation is not yet clear, but current hypotheses will be dis-
cussed in more detail below.
Beyond the obvious agronomic interest in using defoliation as
a means of controlling pollen production, the same approach can
also provide a useful experimental system by which to investi-
gate the process of sex determination in oil palm. In the
context of the studies described here, defoliation can be
dened as the pruning of all expanded leaves at their bases so
as to remove the entire lamina, the unopened leaves in the
spear being cut around 1 m from their base (Durand-Gasselin
et al., 1999). Field observations of sex ratio changes in response
to defoliation have provided clear indications of the chronology
of sex determination and differentiation. The timeline of oil
palm inorescence development, as inferred from the effects
of defoliation, are summarized in Fig. 3, as reproduced from
Hartley (1988) by Durand-Gasselin et al. (1999). It can be
seen that the time intervals between the different stages of inor-
escence development vary according to the age of the palm, in
accordance with the lower rate of leaf emission seen in mature
palms compared with young individuals. One important point
to note is that sex determination is not the only effect of leaf
pruning; the latter treatment also induces inorescence abortion.
However, the two effects occur at different phases of develop-
ment: whereas abortion is mostly observed for inorescences
at stages +4 to +10 (46 months prior to ower maturity),
sex determination changes are typically seen in the region of
stage 20, approx. 22 months before ower maturity in adult
palms (summarized in Durand-Gasselin et al., 1999). As with
most aspects of owering, the exact chronology of these
effects varies according to palm age. Although it can be
Vegetative
meristem
Individualization of
rachis meristem
Individualization of
floral meristem ( )
Floral development
Leaf 20
Leaf 26
Leaf +6
Leaf +15
Leaf +18
Flower maturity
Leaf 40
Initiation of floral bracts
Leaf +2
Initiation of rachis meristem
Leaf 27
Initiation of prophyll and first
peduncular bract
Initiation of rachilla
bract ( )
Leaf 6
Initiation of rachilla
meristem ()
Leaf 2
Individualization of rachilla
meristem ( )
Sex
determination
FI G. 2. Key stages of inorescence development in relation to leaf number.
Adam et al. Environmental regulation of sex determination in oil palm 1531

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promoted articially by leaf pruning, inorescence abortion is in
fact a commonly observed phenomenon in oil palms. Some evi-
dence has been obtained to suggest that inorescence abortion is
sex-dependent, by examining the effects of moderate leaf
pruning (Corley, 1973). In the latter study, no effect was
observed on the sex of inorescences reaching maturity during
the rst 3 months after pruning; however, over the following 6
months, the sex ratio was signicantly lowered. This indicates
that in the specic case of leaf-pruning-related stress at least,
female inorescences undergo a higher rate of abortion than
male inorescences. It should be borne in mind that the more
extreme stress induced by pruning the laminae of all expanded
leaves results in the abortion of inorescences of both sexes.
The ability of oil palms to change the sex of the inores-
cences they produce in response to environmental factors pro-
vides the basis for a useful experimental system to manipulate
reproductive development in palms and study its molecular
determinants. To date, the character of sex determination
per se has not been used by oil palm breeders in genetic
improvement programmes, so nothing is known regarding
the number or type of genetic loci which might be involved
in determining maleness or femaleness. Nevertheless, the
selection of genotypes conferring high oil yield has inevitably
been accompanied by a general increase in sex ratios, which
provides indirect evidence that genetic factors also come into
play. Indeed, with palms of highly selected genotypes
planted in favourable growing conditions, it is commonly
observed that male inorescence production is close to zero
in the rst year or two of owering, thus jeopardizing fruit
production. For this reason, seed batches of high-yielding
genetic crosses are now sometimes supplied mixed with a
small proportion of seeds derived from super-male individ-
uals. The fact that oil palm sex ratios may be enhanced or
reduced depending on genotype, coupled with the fact that a
genetic map has been produced for this species (Billotte
et al., 2001), raises the interesting possibility of identifying
sex determination loci in the future.
ENVI RONMENTAL REGULATI ON OF SEX
DETERMINATI ON WITHIN AND BEYOND
THE ARECACEAE
The ability to vary sexual expression in response to the
environment provides plants with a means of adjusting
resources allocated to ovule and pollen production, thereby
conferring adaptive exibility (Korpelainen, 1998). Gender
plasticity has been observed in plant species belonging to a
wide range of phylogenetic groups. In the case of androdioe-
cious populations of Mercurialis annua (Euphorbiaceae), in
which male and hermaphrodite plants coexist, progeny sex
ratios were found to depend on the density of the parental
plants (Dorken and Pannell, 2008). This phenomenon was
explained in part by the increased pollen production of her-
maphrodite plants when growing at low density, leading to
proportionally fewer male plants in the subsequent progeny.
Other environmental factors that have been shown to affect
sex ratios in plant populations include daylength and light
intensity, as illustrated by the monoecious (or sometime poly-
gamous) species Atriplex halimus (Chenopodiaceae), which
displays increased femaleness under short days and low light
irradiance (Talamali et al., 2001, 2003). In the case of the
sexually dimorphic woody pioneer species Antirhea borbonica
(Rubiaceae), a polliniferous morph was identied and studied
(Litrico et al., 2005). It was found to display considerable
gender plasticity, almost no fruit-bearing female owers
being produced in pioneer populations, whereas in late succes-
sion, high fruit-sets were observed (although they were lower
than for the female morph). In a clonal context, gender plas-
ticity may operate not only in relation to environmental con-
ditions but also in relation to the maturation of the plant, as
seen in the andromonoecious species Pennisetum typhoides
(Poaceae; Sandmeier and Dajoz, 2000) or the monoecious
aquatic Sagittaria sagittifolia (Alismataceae; Dorken and
Barrett, 2003). In the case of the subdioecious species
Urtica dioica (Urticaceae), in which monoecious plants
LEAF POSITION
20 10 0 +10 +20 30 40
Adult palms (two leaves emitted per month)
Young palms (three leaves emitted per month)
22 months 11 months 55 6 months
75 months
LEAF
INITIATION
SEX
DETERMINATION
INFLORESCENCE
ABORTION
FLOWER
MATURITY
FRUIT
MATURITY
LEAF
OPENING
145 months 55 6 months
FI G. 3. Development and sex determination of the oil palm inorescence in relation to palm age [after Hartley (1988), as reproduced by Durand-Gasselin et al.
(1999)].
Adam et al. Environmental regulation of sex determination in oil palm 1532

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coexist with female and male individuals, only the former
were found to display a variable sex ratio, with different
clones of the same genotype varying in the percentage of
male owers produced (Glawe and de Jong, 2005).
Subsequent studies revealed that the maternal parent strongly
contributes to sex ratios, the exact genetic mechanism involved
remaining as yet unresolved (Glawe and de Jong, 2007, 2009).
Although relatively fewdata are available for the palmfamily,
sexual expression has been shown to be inuenced by environ-
mental factors not only in oil palm but also in two different
species of the monoecious Arecoid genus Attalea. In
A. funifera, a study was carried out in Brazil by Voeks (1988)
on the relative abundance of the three different inorescence
types produced: male with staminate owers, bisexual with a
few pistillate and many staminate owers, and female with
pistillate and sterile staminate owers. Although bagging exper-
iments had revealed that autogamy was possible in the bisexual
inorescences, the latter contribute only very weakly to fruit-set,
given their lowabundance at the population level (5 %compared
with 80 % male and 15 % female inorescences). The data
obtained in this study suggested that sexual expression in
A. funifera is related to energetic factors, with palms apparently
producing only male inorescences in the crowded understorey
whereas female expression was observed when they had grown
and entered the energy-rich canopy. Reproductive cost measure-
ments (estimated via dry weights of inorescence and infructes-
cence components) revealed the higher energetic demands of
mature female owers and fruit compared with male owers
(1
.
6 and 13
.
2 versus 1
.
0 in relative units, respectively), with
the production of an initial female inorescence requiring an
energy input estimated as over 10 times the annual budget of
the youngest mature palms. A second species of Attalea,
A. speciosa, was also studied in terms of its gender plasticity
(Barot et al., 2005). In this case, the onset of reproduction and
sex allocation were compared in three contrasting environments:
forest, partially invaded pasture and a pure stand (babassual).
Reproduction was found to start at a smaller size in the pasture
and pure stand compared with forest. Male owering started at
a lower height than female owering in the pasture and pure
stand; however, this was not the case in the forest environment.
Although several hypotheses were raised to explain these data, it
seems probable that the earlier owering observed in more open
environments was made possible by the much greater light (and
therefore energy resources) available to palms in these habitats.
As regards the earlier male owering in pasture/babassual, it was
postulated that the lower levels of stored energy in younger
palms (compared with larger mature palms in the forest) might
be stretched beyond viability by the higher demands of female
owering and fruiting. Thus, the delayed female owering of
mature pasture/babassual palms might be considered as an adap-
tation conferring more efcient energy use and therefore
survival.
At a more general level, although there is some scope for the
optimization of resource allocation to male and female repro-
duction in hermaphrodite species by the modulation of pollen
and ovule development, the greatest exibility is probably
afforded by the monoecious and polygamous conditions,
which allow the adjustment of the relative numbers of special-
ized male and female owers both within the same plant and
between individuals. Within the palm family it seems likely
that the gender plasticity of at least some of the other ve tem-
porally dioecious genera producing unisexual inorescences
(see above) might be subject to environmental regulation.
One intriguing case is that of the Madagascan Marojejya
darianii: a number of specimens of this species were success-
fully grown to maturity on the island of Hawaii, but as yet,
after several years of owering, only male inorescence pro-
duction has been observed (Marcus, 2010). Although environ-
mental factors may play a causal role in cases such as this, it
can also be speculated that endogenous factors within the
plant that change during its life-cycle progression (e.g.
hormone composition), or indeed a combination of the two
elements, may be involved.
MOLECULAR DETERMI NANTS OF FLOWER
AND INFLORESCENCE SEX: CAN WE LEARN
FROM OTHER SPECI ES?
Dioecy and monoecy appear to have arisen on a large number
of occasions during the evolution of owering plants, as
witnessed by their wide and fragmented distribution within
the Angiosperm tree of life. This suggests that the genetic
changes that resulted in the monoecious and dioecious habits
are different for the most part in different evolutionary
lineages (Ainsworth, 2000). Nevertheless, thanks to compara-
tive genomics and evo-devo approaches, it is clear that a wide
range of signalling elements that regulate reproductive devel-
opment are conserved throughout owering plants. An early
indication of this conservation was obtained by comparing
the rst two Angiosperms to have their genomes sequenced,
Arabidopsis thaliana and rice (Izawa et al., 2003). The latter
authors carried out a survey of the structural and functional
conservation of genes between the two model plants, using
owering pathways as an example. This study revealed that a
wide range of owering-related genes are structurally con-
served between monocots and eudicots. Nevertheless, func-
tional divergence appears to have frequently occurred to give
novel regulatory mechanisms which distinguish the two
model species, this degree of divergence providing a glimpse
of the situation in owering plants as a whole. As regards
sex determination, although it seems likely that a generalized
signalling cascade for ower development has been partially
maintained during evolution, the genetic basis of sexual
dimorphism per se is likely to vary considerably. From a
developmental point of view, this hypothesis is supported by
the great variety of ways in which unisexual ower develop-
ment is achieved. Examples of the latter, provided by the
palm family, include non-initiation of the undesired organ
(e.g. the pistil of staminate owers in certain Phytelephas
species), early developmental arrest of the undesired organ
(e.g. the androecium of pistillate owers in Elaeis guineensis)
or incomplete development of the gametophyte (e.g. the
androecium of pistillate owers in Rhapidophyllum hystrix)
(Uhl and Moore, 1977; Adam et al., 2005; Dranseld et al.,
2008). As discussed by Weiblen et al. (2000), dioecy and
monoecy have probably arisen independently a number of
times since the palm family rst originated; for this reason,
the Arecaceae provide an interesting microcosm for studying
the evolution of sexual dimorphism in owering plants as a
whole.
Adam et al. Environmental regulation of sex determination in oil palm 1533

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SEX DETERMI NATI ON MODELS I N
ANGIOSPERMS
Although approx. 10 % of owering plants produce unisexual
owers, relatively few data are available on the genetic basis of
sexual dimorphism, whether in dioecious or in monoecious
plants. Genome sequences have already been obtained for
the dioecious species poplar (Tuskan et al., 2006) and the
trioecious papaya (Ming et al., 2008). In the latter case a
small male-specic region (MSY) was identied on the Y
chromosome and it was proposed that two sex determination
genes controlled the sex determination: a feminizing or
stamen suppressor gene causing stamen abortion before or at
ower inception; and a carpel suppressor gene causing
carpel abortion later in ower development (Ming et al.,
2007). The most extensively studied dioecious model is that
of Silene latifolia, which uses an X/Y-type sex determination
mechanism. For this species, a study was made of the pheno-
typic effects of X-ray-induced deletions (Lebel-Hardenack
et al., 2002), in conjunction with an amplied fragment
length polymorphism approach to identify sex determination
mutants that had lost male traits. This approach provided
data to conrm the classical genetic prediction of three dis-
persed male-determining loci on the Y chromosome, one for
carpel suppression, one for early stamen development and
another for late stamen development. A similar approach was
used by Koizumi et al. (2010). In this case C-ion beam radi-
ation was used to obtain a Y chromosome lesion that resulted
in the production of hermaphrodite owers with a mature
gynoecium. Although the identity of the gene or genes respon-
sible for each of these activities remains to be determined,
complementary studies of sex-specic expression have
enabled the identication of a number of genes of interest
(Robertson et al., 1997; Scutt et al., 2002; Zluvova et al.,
2006) and microdissection techniques have allowed the iso-
lation of Y chromosome-located genes (Deliche`re et al.,
1999; Matsunaga et al., 2003).
Two plant species for which sex determination gene func-
tions have been clearly attributed are melon and maize. In
the case of the polygamous species melon (Cucumis melo),
sex ratios are controlled by allelic compositions at two differ-
ent loci. Boualem et al. (2008) showed that the a allele present
at the androdioecious locus encoded an ethylene biosynthesis
enzyme, CmACS-7, that repressed stamen development in
female owers when active. Martin et al. (2009) later
showed that the transition from male to female owers in
gynoecious lines resulted from the insertion of a transposon
(and consequent DNA methylation changes) in the promoter
of a zinc nger transcription factor gene, CmWIP1.
Expression of CmWIP1, which was abolished in the transpo-
son insertion mutant, led to carpel abortion and also indirectly
repressed the expression of CmACS-7 to allow stamen develop-
ment. Collectively, these data provide an interesting insight
into the sex determination process in the Cucurbitaceae and
also reveal some of the genetic basis of hormone-dependent
sex determination, a common phenomenon in diclinous plant
species.
Another functionally characterized model is that of the
monoecious species maize, which produces morphologically
distinct male and female inorescences on the same plant.
For this species, a number of tasselseed mutants displaying
feminized male owers have been isolated and characterized
and a signalling pathway has emerged (Calderon-Urrea and
Dellaporta, 1999). It was found that the sex determination
genes TASSELSEED1 and TASSELSEED2 are required for
the programmed death of pistil cells in the male ower,
TASSELSEED1 being a putative upstream regulator of
TASSELSEED 2. These two genes were cloned and found to
encode, respectively, a lipoxygenase involved in jasmonic
acid synthesis and a steroid dehydrogenase (DeLong et al.,
1993; Acosta et al., 2009). A third gene, SILKLESS1, of as
yet unknown sequence, blocks TASSELSEED pathway-
induced cell death in the pistil primordia of primary ear
orets. Other studies have implicated the RNA-dependent
RNA polymerase gene RMR6 as a regulator of SILKLESS1
(Parkinson et al., 2007), suggesting crosstalk with epigenetic
regulatory pathways. It is also interesting to note that crosstalk
was observed with the signalling mechanism determining
inorescence branching, itself a sex-linked character (Chuck
et al., 2007). The latter authors demonstrated that the sex
determination gene TASSELSEED4 encoded a micro-RNA
that is able to bind to and regulate the turnover of mRNAs
of the TASSELSEED6 gene related to APETALA2 of
A. thaliana. Further observations by Hultquist and Dorweiler
(2008) led to a wider model which also included the
micro-RNA miR156 and target genes of the
SQUAMOSA-binding protein box (SPB-box) family.
In an evolutionary context, following the discovery of the
tasselseed sex determination pathway in maize, several
studies were performed with the aim of determining whether
all or some of the signalling elements identied might be func-
tionally conserved in other plant groups. Overall, the data
obtained suggest that although orthologues of the
TASSELSEED genes can be easily identied in other species,
their functions probably vary even within the same plant
family. Thus, although the maize pistil abortion mechanism
appears to be conserved in its wild relative Tripsacum dacty-
loides (Li et al., 1997), there is little evidence for conservation
beyond the subfamily Panicoideae of the Poaceae (Zaitchik
et al., 2000; Malcomber and Kellogg, 2006). It is interesting
to note that homologues of TASSELSEED2 identied in
Silene latifolia and A. thaliana displayed male-specic
expression; however, in both cases, this expression was
observed in the tapetum rather than in the gynoecium primor-
dium as in maize (Lebel-Hardenack et al., 1997).
In addition to the tasselseed pathway, maize has also yielded
informative data on the likely role of plant hormones in the
sexual differentiation process. Bensen et al. (1995) character-
ized the ANTHER EAR1 (AN1) gene, mutations of which
result in both reduced plant height and the development of
perfect owers on normally pistillate ears. It was postulated
that the AN1 gene encoded a kaurene synthase A enzyme
involved in gibberellin biosynthesis, which explained the
observed modulation of phenotype severity in response to gib-
berellin application. A further demonstration of the involve-
ment of hormones in the sexual differentiation process in
maize was obtained by Young et al. (2004) and Pineda Rodo
et al. (2008), who demonstrated that increases in biologically
active cytokinin levels achieved by genetic transformation
Adam et al. Environmental regulation of sex determination in oil palm 1534

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led to the abolition of pistil abortion and the appearance of the
tasselseed phenotype.
As regards the question of sex determination in oil palm, the
lack of a widely conserved pathway for this key developmental
process amongst owering plants suggests that at best only
part of the knowledge gained in model plants can be transposed
directly to the Arecaceae. In terms of phylogenetic relationships,
the Poaceae are clearly the closest family for which in-depth
knowledge has been obtained, through studies on maize. The
latter species is therefore a useful starting point from which to
initiate searches for palm orthologues of key genes previously
shown to be involved in sex differentiation, notably those ident-
ied via the tasselseed mutants. The increasing bodyof genomic
sequence data now becoming available for palms, including the
date palm draft sequence (Al-Dous et al., 2011), will prove very
useful in this connection.
TOWARDS A MODEL TO EXPLAIN SEX
DETERMI NATION IN OI L PALM
The fact that oil palm sex determination is a complex process
is amply demonstrated by the complexity of phenological
information obtained from plants growing in the eld.
Although the currently available data do not allow a precise
model to be formulated to explain how oil palm sex determi-
nation is regulated, we can nevertheless identify various
factors of importance and propose a possible framework for
their interactions. At least four different types of factor can
be identied that might participate in sex determination and
differentiation: abiotic factors (e.g. water stress), metabolic
factors (e.g. carbon reserves), hormone status and genetic
factors. The masculinizing effects of drought conditions are
already well known; however, the physiological and genetic
bases of this induced developmental change remain to be elu-
cidated. In one of the earliest publications to mention the
subject of gender plasticity in oil palm, Beirnaert (1935) pos-
tulated that the ratio of carbon assimilation to nitrogen absorp-
tion was the key factor determining the sex ratio. This
hypothesis can be reconciled with the observed effects of
drought if the lowering of photosynthesis in conditions of
water stress is taken into account. Other factors affecting the
metabolic status of palms, including the effects of high-density
planting, fruit pruning and the defoliation of neighbouring
palms, are all compatible with the notion that higher sex
ratios are associated with higher levels of carbon assimilates
(Sparnaaij, 1960). Compared with owering plants as a
whole, oil palm exhibits relatively low phenotypic plasticity
in terms of its overall architecture; transitory carbon reserves
therefore provide a more exible means by which to buffer
sourcesink imbalances associated with climate variability
in this species (Legros et al., 2009c). The latter authors per-
formed a comparative study between oil palms at two different
sites in Indonesia with or without a dry season; data were also
obtained on the effects of fruit pruning and photoperiod vari-
ation (Legros et al., 2009a, b). One of the main elements
revealed by this work was that sourcesink imbalances in
oil palm were buffered principally by uctuations in non-
structural carbohydrates in the stem, which consisted mostly
of glucose and starch. Of these two components, starch was
identied as a major physiological buffer of seasonal
sourcesink imbalances, but glucose was not (Legros et al.,
2009c). Interestingly, however, glucose levels were found to
decrease progressively in response to fruit pruning over the
22-month period of study (Legros et al., 2009b).
As regards the possible involvement of hormones in oil palm
sex determination, some indications were obtained by using the
experimental strategy of applying growth regulators to young
palms grown in large polythene bags (Corley, 1976b). In the
latter study, the auxin NAA (a-naphthyleneacetic acid) was
found to promote female inorescence production; however,
this effect was not observed until 24 months after the end of treat-
ment, whereas sex differentiation was expected to take place
1622 months before anthesis. For this reason, the feminizing
effect was assumed to be indirect. In the case of ethephon, the
predominant effect was to delay owering, albeit with a statisti-
cally insignicant lowering of the sex ratio. Treatment with gib-
berellic acid, however, was shown to signicantly increase the
production of male inorescences and reduce production of
female inorescences within the expected time frame; gibberel-
lins are thus likely to playa role in the oil palmsex determination
process. It is interesting to note that a masculinizing effect of
gibberellic acid treatment has also been observed in cucumber
(Fuchs et al., 1977), whereas in maize, a feminizing effect is
observed with this type of treatment (Dellaporta and
Calderon-Urrea, 1994). These opposing effects observed in
different species no doubt reect the diversity of sex-
determining mechanisms that have evolved in owering
plants, even if some regulatory modules appear to be structurally
conserved. Given the relatively distant phylogenetic relation-
ships between the Arecaceae and the various species for which
Water
availability
Photosynthesis
Starch
Hormone status
Glucose
Genetic factors
Indeterminate
inflorescence
Female
inflorescence
Male
inflorescence
FI G. 4. Possible modes of interaction of factors affecting inorescence sex in
oil palm.
Adam et al. Environmental regulation of sex determination in oil palm 1535

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functional sex determination studies have been performed, it is
clear that prior conceptions need to be avoided when tackling
the complex issue of how this process is regulated in oil palm.
By combining the various data discussed above, a basic fra-
mework to explain the interactions between the various par-
ameters of importance in oil palm sex determination can be
proposed (Fig. 4). The schema shown draws on mostly pheno-
logical and physiological data and suggests points of inter-
action with genetic factors that might be associated with the
contrasting behaviour of palms of different genotype in the
same environmental conditions. A number of questions
remain, regarding notably the modes of interaction between
metabolism, hormone status and the sex determination
process per se. The available evidence, along with general per-
spectives on plant responses to environmental change, point
towards a role for hormones as an intermediary signalling
element between water balance and sex determination, with
metabolic factors possibly acting upstream of the hormone
molecule(s). The model shown is a rst attempt to draw all
these elements together and will now need to be validated,
adjusted and rened by combining new functional genomic
and metabolomic approaches with phenological observations
in the eld. As a rst step in this direction, we are currently
characterizing the transcriptome of the oil palm inorescence,
with a view to identifying points of divergence in the gene
expression patterns associated with male and female develop-
ment. This work will be combined with hormone studies on
the same material, and ultimately the genes of interest display-
ing expression patterns related to male or female differen-
tiation will be studied in a more complex phenological
context. This overall approach should also help to shed light
on events further upstream (i.e. mediating the response to
environmental conditions) and on the role of metabolic
factors in the sex determination process.
CONCLUSI ONS
In this review, we have presented and discussed the wide
ranging but often fragmentary data currently available on the
regulation of sex determination in oil palm. New efforts cur-
rently being devoted to this eld will no doubt shed light on
how oil palm regulates inorescence sex in response to its
environment and reveal to what extent the genetic and physio-
logical elements identied in other species are conserved. Due
to various characteristics that typify palms in general (notably
their large size and long life cycle), research on oil palm ow-
ering is complicated but nevertheless vital to understand the
biology of this important crop plant. The knowledge gained
should also in the longer term improve our understanding of
the evolution of sex determination in the Arecaceae as a whole.
ACKNOWLEDGEMENTS
We gratefully acknowledge nancial support for this work via
a grant from PalmElit SAS and institutional funding from IRD
and Cirad.
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