The origin and mode of function of the Female locus in cucumber

Abstract

Gynoecy in cucumber is controlled by the Female (F) locus that can be modified by other sex determining genes, environmental conditions and plant hormones. As in most of the Cucurbitaceae, the gaseous plant hormone ethylene induces female flower development and thus simulates the activity of the F locus. Monoecious (ff) cucumbers posses a single CsACS1 gene, while gynoecious plants posses an additional female-specific copy, denoted CsACS1G that was mapped to the cucumber Female (F) locus. To study the origin and function of the F locus we compared their genomic structure and investigated their expression pattern and the effect of reducing CsACS1/G transcript level in gynoecious plants. The CsACS1G and CsACS1 genes are identical twin genes except for the distal promoter region that is unique to each gene. The proximal promoter and the distal promoter of CsACS1G contain auxin response regulatory elements (AuxRE) while GA (gibberellic acid) response and repression elements are present in the proximal and distal promoters of both genes. Expression analyses indicated CsACS1/G is expressed in stamen primordia of gynoecious floral buds and that its expression is induced by auxin and repressed by GA. Gynoecious plants with reduced CsACS1/G transcript level present a monoecious phenotype. Altogether our data indicates that the female-specific CsACS1G, and thereby gynoecy in cucumber arose by gene duplication and chromosomal recombination and rearrangement. Furthermore our findings confirm that CsACS1G is indeed the F locus and that one of its activities is to repress stamen development in gynoecious floral buds. INTRODUCTION On monoecious wild type cucumber plant, flowers are developed in a preset developmental sequence along the main stem: a phase of staminate flowers is followed by a mixed phase of staminate and pistillate flowers, and terminated by a phase of pistillate flowers (Shifriss 1961). The nature and length of each phase is genetically determined. Phenotypic expression of sex determining loci is strongly modified by environmental and hormonal factors. Long days, high temperature, and GA promote formation of male flowers, whereas short days, low temperature, ethylene and auxin promote the formation of female flowers (Malepszy and

Full text

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The origin and mode of function of the Female locus in cucumber
1
A. Shiber
1
, R. K. Gaur
2
, R. Rimon-Knopf
1
, A. Zelcer
2
, and T. Trebitsh
1*
1
Department of Life sciences Ben-Gurion University of the Negev, P.O.B 653, Beer-Sheva
84105, Israel
2
Department of Plant Genetics Agricultural Research Organization, Volcani Center, Bet
Dagan 50250, Israel
* Corresponding author e-mail: trebitsh@bgu.ac.il
Keywords: CsACS1/G, Cucumis sativus, ethylene, gynoecy, sex-determination
Abstract
Gynoecy in cucumber is controlled by the Female (F) locus that can be
modified by other sex determining genes, environmental conditions and plant
hormones. As in most of the Cucurbitaceae, the gaseous plant hormone ethylene
induces female flower development and thus simulates the activity of the F locus.
Monoecious (ff) cucumbers posses a single CsACS1 gene, while gynoecious plants
posses an additional female-specific copy, denoted CsACS1G that was mapped to the
cucumber Female (F) locus. To study the origin and function of the F locus we
compared their genomic structure and investigated their expression pattern and the
effect of reducing CsACS1/G transcript level in gynoecious plants. The CsACS1G and
CsACS1 genes are identical twin genes except for the distal promoter region that is
unique to each gene. The proximal promoter and the distal promoter of CsACS1G
contain auxin response regulatory elements (AuxRE) while GA (gibberellic acid)
response and repression elements are present in the proximal and distal promoters of
both genes. Expression analyses indicated CsACS1/G is expressed in stamen
primordia of gynoecious floral buds and that its expression is induced by auxin and
repressed by GA. Gynoecious plants with reduced CsACS1/G transcript level present
a monoecious phenotype. Altogether our data indicates that the female-specific
CsACS1G, and thereby gynoecy in cucumber arose by gene duplication and
chromosomal recombination and rearrangement. Furthermore our findings confirm
that CsACS1G is indeed the F locus and that one of its activities is to repress stamen
development in gynoecious floral buds.
INTRODUCTION
On monoecious wild type cucumber plant, flowers are developed in a preset
developmental sequence along the main stem: a phase of staminate flowers is
followed by a mixed phase of staminate and pistillate flowers, and terminated by a
phase of pistillate flowers (Shifriss 1961). The nature and length of each phase is
genetically determined. Phenotypic expression of sex determining loci is strongly
modified by environmental and hormonal factors. Long days, high temperature, and
GA promote formation of male flowers, whereas short days, low temperature,
ethylene and auxin promote the formation of female flowers (Malepszy and
1
Cucurbitaceae 2008, Proceedings of the IX
th
EUCARPIA meeting on genetics and breeding
of Cucurbitaceae (Pitrat M, ed), INRA, Avignon (France), May 21-24
th
, 2008

264
Niermirowicz-Szczytt 1991; Perl-Treves 1999). Female sex expression is regulated
by the Female (F) locus that interacts with other sex determining loci (Pierce and
Wehner 1990). Extensive research has been conducted to elucidate the molecular
basis of sex determination in cucumber. Cucumber homologues of AGAMOUS (Class
C) are differentially expressed between male and female cucumber flowers but are
not regulated by sex hormones (Perl-Treves 1999; Kater et al. 2001). A Class B gene
was also shown to be involved in developmental arrest of male organs and an
ethylene-induced MADS-box gene (ERAF17) and was suggested to mediate ethylene
induction of female flower formation (Ando et al. 2001; Kater et al. 2001). Genes
involved in ethylene (the main female hormone) synthesis/perception were also
implicated in cucumber floral development (Kamachi and Sakai 1997; Trebitsh et al.
1997; Shiomi et al. 1998; Kahana et al. 1999; Saraf-Levy et al. 2000; Mathooko et al.
2001; Yamasaki et al. 2003). It was proposed that ethylene signals might influence
the product of the M locus and thus inhibit stamen development in cucumber (Saraf-
Levy et al. 2000; Yamasaki et al. 2001). Two ACC synthase genes (ACS), a key
regulatory enzyme in the ethylene biosynthetic pathway were found to involved in
female flower production CsACS2 and CsACS1/G (Trebitsh et al. 1997; Saito et al.
2007). Expression of CsACS2 in individual flowers was suggested to be associated
with differentiation and development of female flowers (Saito et al. 2007) and
CsACS1G was mapped to the F locus (Trebitsh et al. 1997). Monoecious cucumbers
producing mainly male flowers contain a single gene copy (CsACS1), while
gynoecious plants have an additional female-specific copy (CsACS1G). A perfect
correlation (100 %) was found between female phenotypes possessing at least one
dominant F allele and the presence of CsACS1G (Trebitsh et al. 1997).
Identification of CsACS1G as the putative F locus provides an excellent
opportunity to study the molecular mechanisms involved in unisexual flower
development, and the interdependence between developmental cues, hormonal
regulation and gene function.
MATERIALS AND METHODS
Plant material and treatments
Monoecious (MMff) and gynoecious (MMFF) near-isogenic cucumber lines
(Cucumis sativus L. var sativus ‘Beit Alpha’) were used to isolate and examine gene
expression. These lines were received from HaZera Seed Company (Israel). Seed of
the gynoecious cucumber used for reducing CsACS1/G transcript level (C. sativus L.
var sativus ‘Ilan’; MMFF) were received from Zeraim Gedera Ltd. (Gedera, Israel).
Seeds were germinated and grown in a mixture of peat and vermiculite (1:1). Plants
were grown in a growth chamber under a under a 16-h photoperiod and 27±1
o
C.
For regulation of gene expression, in vitro shoot apices were harvested when
the second leaf was fully expanded. Excised apices were treated for 6 hrs as
previously (Trebitsh et al. 1997) with or without 100 mM IAA or 500 mM GA
4+7
(Duchefa Biochemie, Haarlem, The Netherlands).
RNA analysis
RNA extraction and RNA blot hybridization analysis were performed as
previously with probes derived from CsACS1/G or BCAT genes as indicated in the
figures (Rimon Knopf and Trebitsh 2006; Barak and Trebitsh 2007).

265
Transient assays
Rapid in vivo analysis of CsACS1/G promoter activity was performed by
transient expression assays following published procedures (Yang et al. 2000). Binary
vector pCAMBIA 1303 (CSIRO, Australia) that contains GUS gene driven by CaMV
35S or CsACS1/G promoter or a promoterless GUS were constructed. Agrobacterium
tumefaciens EHA 105 cells, carrying the appropriate plasmid construct were
infiltrated into fully expanded Nicotiana benthamiana leaves still attached to the
plant. Leaves were infiltrated with 100µl of bacterial suspension and after 72 hs were
examined for GUS activity according to standard procedures (Wroblewski et al.
2005).
Cucumber stable transformation
For stable gene transformation of cucumber plants we used an established
transformation method (Gal-On et al. 2005). Cotyledon explants were transformed
with Agrobacterium tumefaciens EHA 105 harboring ca. 300bp of the 5’UTR-N-
terminal of CsACS1/G in an intron-containing hairpin transformation construct made
by using the pHANNIBAL/pART27 system (Gleave 1992; Wesley et al. 2001).
Following Agrobacterium + explant co-cultivation, adventitious transgenic shoots
were induced in regeneration medium under stringent selection (MS medium /3 %
sucrose /2 mg/l benzylaminopurine/100 mg/l kanamycin /250 mg/l claforan). The
transgenic nature of rooted shoots was confirmed by PCR, and young plants were
transferred to the growth chamber (as above) for phenotyping and seed production.
RESULTS AND DISCUSSION
CsACS1 and CsACS1G structure and expression
To study the origin and function of the CsACS1G we isolated the full-length
genomic sequence of CsACS1 from monoecious plants and of CsACS1 and CsACS1G
from gynoecious plants (Rimon Knopf and Trebitsh 2006). We found that the identity
of the non sex-specific CsACS1 and the gynoecious sex-specific CsACS1G extends
from the 3’UTR to the proximal promoter region indicating that the duplication of
CsACS1 occurred relatively recently in evolutionary terms (Fig. 1). We further found
that the distal promoter region of CsACS1G is highly similar to the last two exons of a
branched-chain amino acid transaminase (BCAT) gene that might constitute an open
reading frame (ORF) (Rimon Knopf and Trebitsh 2006). To examine whether this
putative ORF is transcribed we performed RNA blot hybridization analysis
comparing monoecious and gynoecious plants and using the last two exons of the
BCAT gene (exon 9, 10) as a probe. If the putative ORF is transcribed we expected to
detect two transcripts in gynoecious plant: a high molecular transcript for BCAT and a
low molecular one corresponding to the putative ORF. The results show that only one
transcript is detected with this probe in both gynoecious and monoecious plants (Fig.
2). The transcript size is the same as the transcript detected by exon 8 of the BCAT
gene, an exon that is not present in the distal promoter of CsACS1G (Fig. 2). We
conclude that putative ORF is not active thereby ruling out the possibility that its
product may be the F locus.

266
Figure 1. Location of regulatory elements within the promoters of the CsACS1 and
CsACS1G genes. The identical sequences in both genes are marked as CsACS1/G - a
black line is the proximal promoter, 5’UTR is an open box, and downstream is an
open box with bold outline. 1622 bp of the unique distal promoter of CsACS1G and
CsACS1 are shown as open box and hatched box, respectively.
Figure 2. Transcript abundance of the BCAT gene in monoecious (M) and gynoecious
(G) cucumber plant apices. RNA blots were probed with BCAT Exon 8 or with Exon
9 and 10 that are present in the distal CsACS1G.
Figure 3. Expression of CsACS1/G is induced by auxin (0.1mM) and repressed by
GA
4+7
(0.5mM) in monoecious (M) and gynoecious (G) cucumber plant apices. RNA
blots were probed with CsACS1/G transcript
Comparative computerized promoter analysis between the promoters of the
twin genes suggested the presence of both similar and unique putative cis-acting
regulatory elements in the two promoters (http://www.dna.affrc.go.jp/PLACE/). The
proximal promoter that is identical in both genes, as well as the distal promoter that is
unique to each genes contain elements for repression of GA response (Fig. 1). On the

267
other hand, auxin responsive elements (AuxRE) are present in the proximal promoter
and in the distal promoter of CsACS1G (Mibus and Tatlioglu 2004; Rimon Knopf and
Trebitsh 2006) (Fig. 1). The response of CsACS1/G to auxin and GA was examined in
plant growth apices treated for 6 hs (Fig. 3). As shown previously, higher transcript
level is observed in gynoecious plants (CsACS1/G transcript) than in monoecious
(CsACS1 transcript) and expression of CsACS1 or CsACS1/G is induced by auxin in
both lines (Trebitsh et al. 1997; Rimon Knopf and Trebitsh 2006). Treatments with
GA reduced the expression of CsACS1/G in both genotypes (Fig. 3). This raises the
possibility that in monoecious plants treatment with auxin induces the expression of
CsACS1 leading to higher ethylene production and to female flower production. This
effect may involve the induction of the ethylene-inducible CsACS2 that was shown to
be expressed in pistil primordia of female flowers developing on monoecious plants
(Saito et al. 2007). The finding that GA reduced the expression of CsACS1/G suggests
that the masculinization effect of GA on gynoecious plants may be by down-
regulating the expression of CsACS1/G.
CsACS1/G functional activity
Transient expression assay is a well-tried procedure to rapidly evaluate plant
promoters and transcription factors in vivo (Yang et al. 2000). Agrobacterium cells,
carrying plasmid constructs were infiltrated into N. benthamiana leaves in planta to
examine the activity of the proximal promoter that is identical in both genes (Figs. 1,
4). Activity of GUS driven by the proximal promoter was comparable to its activity
driven by the constitutive promoter CaMV 35S. Thus the proximal promoter is
sufficient to induce transcription of CsACS1/G (Fig. 4). Therefore, any differential
expression between the twin genes is likely to originate from unique regulatory
elements (induction or repression) residing in the distal promoter of the twin genes. It
is noteworthy that the distal promoter of CsACS1G contains a CArG domain that can
bind MADS-domain transcription factors. In gynoecious plants, at the sex
determination stage CsACS1/G is expressed in the stamen primordia (data not
shown). Thus, CsACS1G may be regulated by a homeotic gene, such as CUM1,
leading to repression of stamen primordia at the critical developmental stage (Kater et
al. 2001; Bai et al. 2004)
.
Figure 4. CsACS1/G promoter activity in N. benthamiana leaves. GUS expression
was examined in leaves infiltrated with Agrobacterium cells harboring either a
promoterless GUS (Control), one driven by CaMV 35S (35S) or by the proximal
promoter of CsACS1/G (Proximal CsACS1/G)
To test the function of CsACS1G we generated seventeen independent
gynoecious cucumber plants (‘Ilan’) in which the transcript level of CsACS1/G was

268
reduced by RNA interference technique. All lines exhibited various degrees of
monoecious phenotype and on average the appearance of the first female flower was
delayed to the 13
th
node while the transition to female phase was postponed to the 18
th
node (Fig. 5).
Figure 5. The effect of CsACS1/G silencing on floral sex phenotype of gynoecious
cucumber. Gynoecious cucumbers (A) transformed with a CsACS1/G silencing vector
(B) show a monoecious phenotype. An arrow marks female flowers
CONCLUSIONS
Taken together our data indicate that an event of gene duplication and an inter-
or intrachromosomal recombination and rearrangement, between a BCAT gene and
CsACS1, in the monoecious cucumber, gave rise to CsACS1G and gynoecy. The
source of femaleness in the majority of cucumber cultivars, is derived from the
Japanese cultivar Shogoin female PI 220860 (Shifriss 1961; Pierce and Wehner
1990). Hence, CsACS1G is a unique example of a gene duplication detected in its
early evolutionary stage and propagated by extensive breeding programs due to its
agricultural benefit. Since the proximal promoter of CsACS1/G is active by itself, any
differential temporal and spatial expression pattern detected between the two genes is
expected to originate from regulatory elements residing in the unique distal promoter
of the genes. Furthermore, CsACS1/G is regulated antagonistically by feminizing and
masculinizing plant growth regulators designating it as the central gene in floral sex
expression. Reducing the level of CsACS1/G transcripts in gynoecious (FF)
cucumbers resulted in a monoecious phenotype, such as those carrying CsACS1 only

269
(ff). We postulate that CsACS1 is involved in developmental processes other than sex
determination but that in response to hormonal treatments may also participate in
floral sex expression (Saito et al. 2005). Finally, based on our results we suggest that
CsACS1G is the F locus that can be modulated by both feminizing and masculinizing
hormones and it functions by suppressing stamen primordia development and
allowing for female flower development and gynoecious phenotype.
ACKNOWLEDGMENT
We thank CSIRO (Canberra, Australia) and Andrew P Gleave (The
Horticulture and Food Research Institute of New Zealand Ltd. Auckland, New
Zealand) for providing pHANNIBAL and pART27 plasmids. This work was
supported by the Chief Scientist of the Israeli Ministry of Agriculture and Rural
Development (Project 854-0488-04).
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