Plant Signaling & Behavior

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Why is ethylene involved in selective promotion of

female flower development in cucumber?

Jin-Jing Sun, Feng Li, Xia Li, Xiao-Chuan Liu, Guang-Yuan Rao, Jing-Chu Luo,
Dong-Hui Wang, Zhi-Hong Xu & Shu-Nong Bai

To cite this article: Jin-Jing Sun, Feng Li, Xia Li, Xiao-Chuan Liu, Guang-Yuan Rao, Jing-Chu
Luo, Dong-Hui Wang, Zhi-Hong Xu & Shu-Nong Bai (2010) Why is ethylene involved in selective
promotion of female flower development in cucumber?, Plant Signaling & Behavior, 5:8, 1052-1056,
DOI: 10.4161/psb.5.8.12411

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Published online: 01 Aug 2010.

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 article addendum

Plant Signaling & Behavior 5:8, 1052-1056; August 2010; © 2010 Landes Bioscience

Why is ethylene involved in selective promotion of female flower

development in cucumber?
Jin-Jing Sun, Feng Li, Xia Li, Xiao-Chuan Liu, Guang-Yuan Rao, Jing-Chu Luo, Dong-Hui Wang, Zhi-Hong Xu and Shu-Nong
Bai*
PKU-Yale Joint Research Center of Agricultural and Plant Molecular Biology; National Key Laboratory of Protein Engineering and Plant Gene Engineering;
College of Life Sciences; Peking University; Beijing, China; and The National Center of Plant Gene Research; Beijing, China

O ur recent work by Wang et al.

Key words: ethylene, F gene, M gene,
microRNA, unisexual flower
Submitted: 05/17/10
Accepted: 05/18/10
Previously published online:
www.landesbioscience.com/journals/psb/
article/12411
*Correspondence to: Shu-Nong Bai;
Email: shunongb@pku.edu.cn
Addendum to: Wang DH, Li F, Duan QH, Han T,
Xu ZH, Bai SN. Ethylene perception is involved
in female cucumber flower development.
Plant J 2010; 61:862–72; PMID: 20030751; DOI:
10.1111/j.1365-313X.2009.04114.x.
(2010), together with previous
studies published in the last decade,
have provided evidence suggesting a
link between ethylene signaling and pri-
mordial anther specific DNA damage in
female cucumber flowers. These find-
ings explained ethylene promotion of
female flower by ethylene inhibition of
stamen development. However, it is not
determined if ethylene promotes carpel
development. In addition, an explana-
tion of why the naturally occurring gas
ethylene was selected to be involved in
such developmental events remains elu-
sive. In this study, we carried out a phylo-
genetic analysis of cucumber ACS genes,
and analyzed the expression levels of
some pre-miRNAs in male, female and
hermaphrodite flowers. We found the
M gene might have evolved prior to, or
“co-opted” into unisexual flower devel-
opment before the F gene, and miRNA
might be involved in carpel development
regulation. Based on these observations,
we propose a new hypothesis to explain
why ethylene was selected to be involved
in the evolution of the unisexual cucum-
ber flower.
The unisexual flower is one of many
interesting innovations to promote cross-
pollination in plants. Morphogenetic
paths leading to unisexual flowers are
known to vary from plant to plant,1 how-
ever, research clearly demonstrates that
phytohormones are the most important
players in unisexual flower development
in Cucumis and other angiosperms.2
Cucumber is considered a model plant
to study ethylene promotion of female
flower in unisexual flower development.3,4
During the last decade, we observed that
inappropriate sexual organs in cucumber
flowers diverge from normal morphoge-
netic paths at stage 6.5 Although DNA
damage was specifically detected at stage
7 primordial anthers in female flowers,6
the inappropriate sexual organs remained
viable through anthesis.6,7 To examine
whether the primordial specific DNA
damage is induced by ethylene, we have
constructed transgenic Arabidopsis con-
taining an AP3 promoter driven over-
expression of the CsACO2 gene, which
encodes a key enzyme for cucumber eth-
ylene synthesis, and found the stamen
development in transgenic Arabidopsis is
significantly affected to the extent that
some of them mimicked the inappropri-
ate stamen in female cucumber flowers.8
We recently reported our findings that
ethylene perception is involved in stamen
development inhibition in female cucum-
ber flowers via induction of DNA dam-
age.9 All of these results taken together
provided a foundation for us to explain
how ethylene selectively inhibits stamen
development in female cucumber flowers.
However, we have not been able to
determine if ethylene promotes carpel
development. If we can answer this ques-
tion, we are closer to understanding the
whole mechanism of “ethylene promotion
of female flower development in cucum-
ber”. Furthermore, as both F and M genes,
the key genes determine sex phenotypes,
encode CsACS,10-13 it was highly curious

1052 Plant Signaling & Behavior Volume 5 Issue 8

 article addendum
for us to ask why it is ethylene, a univer-
sally existing phytohormone, has been
evolutionarily favored by natural selection
to selectively promote of female flower
development in cucumber? Additionally,
and more specially, why are two ACS
genes required for the process?
We took advantage of the recently
completed cucumber genome sequence,14
and carried out two analyses to address
these questions.
In the present study, we phylogeneti-
cally analyzed the available plant ACS
genes in all accessible databases. According
to Zhong-Hua Zhang in Huang’s cucum-
ber genome sequencing group, eight genes
encode ACS proteins in the cucumber
genome (personal communication). Four
genes have been previously reported with
access numbers of BAA33374 (CsACS1),
BAA33375 (CsACS), BAF79596
(CsACS2, the M gene. An additional access
of ACT78959 is recently identified as loss
of function mutation of the M gene12),
and ABI33818 (CsACS1G, the F gene).
In the cucumber genome sequence data-
base managed by Huang’s research group,
there are additional ACS genes identified
as Csa011441, Csa004048, Csa017570
and Csa018243. Figure 1 indicates that
both F and M genes evolved from a clade
including Arabidopsis ACS7.15 It is inter-
esting that following divergence between
the two clades represented by Arabidopsis
ACS7 and ACS10 (indicated by dark red
bars), the M gene is shown to emerged
after six divergence events (indicated by
the bars labeled in red, brown, yellow,
light green, green and light blue), and it
is suggested the F gene emerged follow-
ing 10 divergences (indicated by the bars
labeled in red, brown, yellow, light green,
green, light blue, blue, dark blue, purple
and light purple). This result suggests that
the M gene is ancestral to the F gene, or
evolved faster than the F gene during the
evolutionary process, consistent with a
proposal that a duplication of the F gene
is a more recent evolutionary event.11
Secondly, we carried out quantita-
tive RT-PCR analysis of pre-microRNA
(pre-miR) predicted with two criteria,
sequence similarity and secondary struc-
ture characteristics, in male, female (with
monoecious line Zhongnong 15) and
hermaphrodite (with hermaphrodite line
www.landesbioscience.com Plant Signaling & Behavior 1053
H34) floral buds at stage 9. The original
purpose of this experiment was to verify
the presence of the predicted pre-miRs
(Liu et al. unpublished data). While we
indeed detected expression of some pre-
dicted pre-miRs, we were surprised to
find expression of pre-miR319/159, 171,
as well as 396, 156 and 166, showed a
carpel dependent pattern (Fig. 2). In
these pre-miRs, expression levels were sig-
nificantly lower in male floral buds than
female and hermaphrodite floral buds.
Although we have not examined mature
miRs, these carpel-dependent expression
patterns of pre-miRs suggest miR might
play a role in cucumber unisexual flower
development.
To date, evidence for an association
between microRNA expression and car-
pel development is unavailable. However,
it was reported that when downregulat-
ing the TCP gene family expression via
constitutively expressing gene specific
microRNA, Arabidopsis leaves continued
to grow and increase in size.16 Some TCP
genes are the targets of miR319,17 petal
and stamen development are both affected
by miR319 regulated TCP genes.18
Therefore, low pre-miR expression, par-
ticularly pre-miR319; detected in male
floral buds, imply high TCP gene expres-
sion. If this is the case, and if the regula-
tory mechanism found in Arabidopsis leaf
development is applicable to cucumber
carpel development, miR such as miR319
might be involved in the precocious inhi-
bition of ovary development, as observed
previously,5 through regulating TCP and/
or other transcriptional factor expression.
The above analyses enabled us to pro-
pose a new hypothesis, which may be
called the “miR initiative”, to explain
why ethylene is involved in the selective
promotion of female flower development
(Fig. 3).
In the “miR initiative” hypothesis, we
first presume that unisexual cucumber
flowers were derived from a hermaphrodite
ancestor, according to Barrett.19,20 Then
we hypothesize that due to unknown envi-
ronmental conditions, expression of miRs
was altered, and as a result, expression of
miR target genes, such as TCP, were not
downregulated properly. It is possible due
to evidence demonstrating that the miR is
sensitive to environmental change.21,22 As
article addendum
proposed above, high-level TCP expres-
sion might precociously inhibit ovary
development. Because the environmen-
tally-induced alteration of miR expression
is likely an occasional event, it might occur
only in individual floral buds and there-
fore have generated ancient andromon-
oecious cucumber plants. Such ancient
andromonoecious plants would result in
two opposite effects: one reduction in the
number of offspring caused by the inhibi-
tion of ovary development; and second an
opportunity for cross-pollination.
Although cross-pollination has benefit
for plants to adapt constantly changing
environment, the production of viable off-
spring is one tenet for species survival and
adaptation. If the situation of above envi-
ronment-dependent andromonoecy went
too far for the specie to survive, plants
must do something to protect themselves.
One possible event was the evolution of a
mechanism to rescue ovary development.
De Martinis and Mariani reported that
ethylene benefits ovule development in
tobacco.23 Therefore we hypothesize that
the M gene diverged and co-opted into
this mechanism, resulting in monoecious
(with genotype of MM--). The carpel-
specific expression pattern of the M gene
(CsACS2), as well as its ortholog in melon
A gene (CmACS7),24 is consistent with this
hypothesis. If this scenario is true, eth-
ylene inhibition of stamen development
should be understood as a side effect of the
M gene co-option. Based on our current
knowledge regarding unisexual cucumber
flower development, other modifications
following the M gene co-option might
have occurred during evolution, including
resource allocation between stamens and
carpels during development, and natural
selection of CsETR1 organ-specific down-
regulation of and/or other ethylene signal-
ing components in stamens.
Research has shown that the cucum-
ber m gene is a loss-of-function mutation
of the M gene.12,13 Therefore, we specu-
late that the present andromonoecious
genotype resulted from reverted point
mutations from the monoecious M gene.
Accordingly, we might discover ancient
andromonoecious plants with a genotype
lacking the M gene in wild populations
that are related to modern day cucubits in
the cucumber origin area.
 Figure 1. Phylogenetic analysis of the
ACS family. Protein sequence of Cucumis
sativus ABI33818 was used to search for ACS
homologs in available genome sequences
of all organisms (www.ncbi.nlm.nih.gov/
BLAST/) and in the plant transcript database
of Physcomitrella patens and Selaginella
moellendorfii (sgdb.cbi.pku.edu.cn) us-
ing both BLASTP programs. The multiple
sequence alignment of protein sequence was
performed using ClustalX (Plate-Forme de
Bio-Informatique, Illkirch Cedex, France). The
most conservative regions were aligned and
used to perform neighbor joining analyses
with the pairwise deletion option, Pois-
son correction model, and 1,000 bootstrap
replicates test in MEGA version 4.1 [Kumar S,
Dudley J, Nei M and Tamura K (2008); www.
megasoftware.net]. The single sequence of
Pp gw1.C.2350048 was treated as an out-
group sequences. Pt indicates proteins from
poplar (Populus trichocarpa), Vv (Vitis vinifera);
Zm (Zea mays); Cm (Cucumis melo); Os (Oryza
sativa); Picea (Picea engelmannii x P. glauca);
Sm (Selaginellae moellendorfii); Pp (Physcomi-
trella patens); Osj and Osi here only refer
separately to Oryza sativa japonica group
and O. sativa indica group; Pis refers to Pinus
sylvestris and Pit to P. taeda. The red square
filled with light green indicates the protein
encoded by the cucumber M gene (CsACS2)
and its mutated (m) sequence. The red square
filled with light yellow indicates the protein
encoded by cucumber F gene. Blue squares
indicate the proteins encoded by the remain-
ing cucumber ACS genes. The pink ovals
highlight the Arabidopsis proteins encoded
by the ACS genes for reference. Colored bars
indicate the predicted divergence events pre-
dicted (for details see the text).

Although the emergence of monoe-

cious cucumbers could solve the problem
of offspring reduction caused by environ-
ment-dependent andromonoecious plants,
and ensured cross-pollination, the ratio of
male and female flowers was still strongly
affected by the environment.3 This ren-
dered an opportunity for a new evolu-
tionary event, the F gene duplication, as
suggested by Knopf and Trebitsh11 (also
see the phylogenetic analysis in Fig. 1).
Additional copies of CsACS1G, the F gene,
would increase the ethylene production
and consequently the promotion of ovary
development and inhibition of stamen
development. The gynoecious (MMFF)
genotype resulted in the benefits of both
offspring production and cross-pollina-
tion under endogenous control.
As there is no reproduction barrier
among the different cucumber genotypes,

1054 Plant Signaling & Behavior Volume 5 Issue 8

 Figure 2. Expression levels of selected predicted cucumber pre-miRs. The predicted 21 pre-miRs expression levels were examined because the miR
homologs were reported expressed in other plant species. The pre-miR396a, pre-miR156b, pre-miR319a, pre-miR171b, pre-miR166a exhibited sig-
nificant differences in their expression levels between male flowers in the absence of carpel development, female and hermaphrodite flowers, which
have normal carpel development. These results suggest that the expression levels of the pre-miRs may correlate with the carpel development.

Figure 3. Hypothetical model of unisexual flower evolution in cucumber. Our model hypothesizes that unisexual cucumber flowers evolved from a
hermaphrodite ancestor (gray-lined box). The first event during the evolutionary process might be the miR-mediated arrest of ovary development
(see text). This event resulted in ancient environment-dependent andromonoecy (gray-lined box). To ensure ovary development for seed-set, M gene
was co-opted, which resulted in the side effect of stamen development inhibition to maintain cross-pollination. The co-option of the M gene gener-
ated the monoecious genotype (light yellow filled box). The loss-of-function m gene was considered a reverted point mutation, which generated the
present andromonoecious genotype (light yellow filled box). The F gene was further co-opted, possibly by gene duplication, and generated a gynoe-
cious genotype (light yellow filled box). These three genotypes were considered as core genotypes. The recombination of these three genotypes can
generate various sex phenotypes.

www.landesbioscience.com Plant Signaling & Behavior 1055


at least among those have been described,3 it
is expected that the wide range of diverged
unisexual flower types can be derived from
the crosses and recombinational events
among the three core genotypes (Fig. 3).
To us, the above hypothesis is really
exciting, not only because it present a
reasonable explanation for why gaseous
ethylene is involved in selective promo-
tion of female flowers, but also explains
the origin of male flowers. Furthermore,
this hypothesis provides tremendous new
opportunities to explore the molecular
mechanisms underlying unisexual flower
development. For example, are miR and
TCP genes involved in ovary develop-
ment, and does ethylene promote ovary
development, and if so, does it act through
ethylene regulation of miR or directly via
TCP? However, currently, the greatest
challenge preventing progress is cucumber
transformation. If we can overcome the
obstacles to cucumber transformation in
the near future, we will make substantial
advancements in elucidating the molecu-
lar mechanism of the origins of unisexual
cucumber flowers!
Acknowledgements
We thank San-Wen Huang and his team
(IVF, CAAS) for their help to access the
cucumber genome database; Run Cai
and Jin-Song Pan at Shanghai Jiaotong
University for providing H34 seeds; An
-Min Lu at the Institute of Botany, CAS,
for his help in providing systematic infor-
mation about cucmber relatives; Frantisek
Baluska for her invitation and encourage-
ment of providing Addendum Article in
such a hypothesis style.
1056 Plant Signaling & Behavior Volume 5 Issue 8
13. Li Z, et al. Molecular isolation of the M gene sug-
References
gests that a conserved-residue conversion induces the
1. Dellaporta SL, Calderon-Urrea A. Sex determination
in flowering plants. Plant Cell 1993; 5:1241-51.
formation of bisexual flowers in Cucumber plants.
Genetics 2009; 182:1381-5.
2. Ainsworth C. (ed) Sex Determination in Plants. 14. Huang S, et al. The genome of the cucumber,
BIOS Scientific Publishers Ltd., Oxford (GB) 1999. Cucumis sativus L. Nat Genet 2009; 41:1275-81.
3. Perl-Treves R. Male to female conversion along the
cucumber shoot: approaches to, studying sex genes
15. Yamagami T, et al. Biochemical diversity among
the 1-amino-cyclopropane- 1-carboxylate synthase
and floral development in Cucumis sativus. In: Sex isozymes encoded by the Arabidopsis gene family. J
Determination in, Plants, (Ainsworth CC, ed) BIOS Biol Chem 2003; 278:49102-12.
Scientific Publishers Ltd., Oxford (GB) 1999; 189- 16. Efroni I, Blum E, Goldshmidt A, Eshed Y. A pro-
215. tracted and dynamic maturation schedule under-
4. Perl-Treves R, Rajagopalan PA. Close, yet separate: lies Arabidopsis leaf development. Plant Cell 2008;
patterns of male and female floral development in 20:2293-306.
monecious species. In: Ainsworth C. (ed). Flower 17. Palatnik JF, et al. Control of leaf morphogenesis by
development and manipulation. Blackwell Publisher microRNAs. Nature 2003; 425:257-63.
2006; 117-46.
18. Nag A, King S, Jack T. miR319a targeting of
5. Bai SL, et al. Developmental analyses reveal early
arrests of the spore-bearing parts of reproductive
TCP4 is critical for petal growth and development
in Arabidopsis. Proc Natl Acad Sci USA 2009;
organs in unisexual flowers of cucumber (Cucumis 106:22534-9.
sativus L.). Planta 2004; 220:230-40.
19. Barrett SC. The evolution of plant sexual diversity.
6. Hao YJ, et al. DNA damage in the early primordial
anther is closely correlated with stamen arrest in the
Nat Rev Genet 2002; 3:274-84.
20. Barrett SC. Understanding plant reproductive diver-
female flower of cucumber (Cucumis sativus L.).
sity. Philos Trans R Soc Lond B Biol Sci 2010;
Planta 2003; 217:888-95.
365:99-109.
7. Yang LL, et al. Carpel of cucumber (Cucumis sativus.
L) male flowers maintains early primordia character-
21. Sunkar R, Zhu JK. Novel and stress-regulated
microRNAs and other small RNAs from Arabidopsis.
istics during organ development. Chinese Sci Bull
Plant Cell 2004; 16:2001-19.
2000; 45:729-33.
8. Duan QH, Wang DH, Xu ZH, Bai SN. Stamen
22. Liu HH, Tian X, Li YJ, Wu CA, Zheng CC.
Microarray-based analysis of stress-regulated microR-
development in Arabidopsis is arrested by organ-spe-
NAs in Arabidopsis thaliana. RNA 2008; 14:836-43.
cific overexpression of a cucumber ethylene synthesis
23. De Martinis D, Mariani C. Silencing gene expression
gene CsACO2. Planta 2008; 228:537-43.
9. Wang DH, et al. Ethylene perception is involved in
of the ethylene-forming enzyme results in a revers-
ible inhibition of ovule development in transgenic
female cucumber flower development. Plant J 2009;
tobacco plants. Plant Cell 1999; 11:1061-72.
2010; 61:862-72.
24. Boualem A, et al. A conserved mutation in an ethyl-
10. Mibus H, Tatlioglu T. Molecular characterization
and isolation of the F/f gene for femaleness in cucum-
ene biosynthesis enzyme leads to andromonoecy in
melons. Science 2008; 321:836-8.
ber (Cucumis sativus L.). Theor Appl Genet 2004;
109:1669-76.
11. Knopf RR, Trebitsh T. The female-specific
Cs-ACS1G gene of cucumber. A case of gene duplica-
tion and recombination between the non-sex-specific
1-aminocyclopropane-1-carboxylate synthase gene
and a branched-chain amino acid transaminase gene.
Plant Cell Physiol 2006; 47:1217-28.
12. Boualem A, et al. A conserved ethylene biosynthesis
enzyme leads to andromonoecy in two cucumis spe-
cies. PLoS One 2009; 4:6144.