Delayed Flowering in Pineapples (Ananas comosus (L.) Merr.

) Caused
by Co-Suppression of the ACACS2 Gene
Yuri Trusov and Jose Ramon Botella
Plant Genetic Engineering Laboratory
Department of Botany
School of Integrative Biology
University of Queensland
Brisbane 4072
Australia
Keywords: flowering control, transgenic pineapple, ethylene
Abstract
Natural flower induction is a major pineapple industry problem. It usually
occurs when shortening days and low temperatures give raise to increased ethylene
production in the leaf tissue and plant stem apex which in turn stimulates flowering.
Natural flowering fruit matures 4 to 6 weeks ahead of the normal summer harvest
resulting in the need for extra harvest passes and considerable yield losses. Ethylene is
produced through the sequential action of ACC synthase and ACC oxidase. Our team
has cloned an ACC synthase gene from pineapple (ACACS2), which is expressed in
meristems and activated under the environmental conditions that induce flowering in
nature. Genetic constructs have been produced containing ACACS2 in sense orienta-
tion to induce silencing of the host gene in the plant by co-suppression mechanisms.
Two independent lines of transgenic plants have been produced and field trials have
been conducted in Queensland for four years in order to study the characteristics of
the transgenic lines. We have identified a group of transgenic plants demonstrating
inherited flowering delay and confirmed co-suppression of the ACACS2 gene due to
methylation.
INTRODUCTION
Flowering is one of the most fundamental processes in plant ontogeny,
representing transition from vegetative growth to generative development resulting in
reproduction. It starts with alteration of the apical meristem identity so that it produces
flowers instead of leaves. This transition could be initiated by a wide diversity of
endogenous as well as exogenous factors, the majority of which however, act through a
limited set of specific plant hormones.
Onset of flowering in pineapples is marked by the appearance of a small red
inflorescence in the center of the plant rosette and vibrant red coloration in the base of the
youngest (smallest and located around the central meristem) leaves. Flowering correlates
with plant age and size (Vieira et al., 1983), however, could be triggered in immature
plants by environmental stresses (Friend and Lydon, 1979; Reinhardt et al., 1986;
Bartholomew, 1987; Min and Bartholomew, 1996). In pineapple agronomy, synchronizing
the flowering of plants on a plantation has a critical importance, because of the strong
dependence of fruit ripening on flowering time and the non climacteric nature of the
species. To synchronize flowering, pineapple growers usually select planting material
(pineapple slips) by size/weight (Reinhardt and Medina, 1992) and once plants reach
maturity, (usually a year after planting), treat them with a number of flowering inducing
agents (Bartholomew, 1977; Reid and Wu, 1991). Nevertheless, a certain portion of the
crop (ranging from 5 to 30 percent) manages to flower ahead of schedule. This
phenomenon is known as natural flowering or environmental induction (Min and
Bartholomew, 1996) and is a highly undesirable characteristic of pineapples grown
worldwide making cultivation more laborious and causing significant harvest losses (Min
and Bartholomew, 1996). One obvious solution to this problem is to postpone or prevent
the innate flowering in general.

Proc. V th Intl. Pineapple Symp.

Ed. P.H. Joubert 29
Acta Hort. 702, ISHS 2006
 In pineapples, unlike many other plant species, flowering can be induced by the
gaseous plant hormone ethylene. It has been shown that prior to inflorescence emergence,
the leaf basal-white tissue produces ethylene (Bartholomew, 1977; Min and Bartholomew,
1996). Use of ethylene and ethylene releasing chemicals like ethephon [(2-chloro-
ethyl)phosphonic acid] or etacelasil [2-chloroethyltris(2-methoxyethoxy)silane] has
become a common practice for flowering induction among pineapple growers (Randhawa
et al., 1970; Reid and Wu, 1991; Manica et al., 1994). Auxin can also induce flowering in
pineapple through initiation of ethylene production (Burg and Burg, 1966). Despite
existence of ethylene biosynthesis inhibitors and chemicals disrupting ethylene signaling,
attempts to arrest or at least delay unwanted flowering in pineapple with these agents still
had low success (Min and Bartholomew, 1996; Kuan et al., 2005).
The pathway for ethylene production in plants was established by Adams and
Yang (1979). The key enzyme of this pathway is 1-amino-cyclopropane-1-carboxylate
synthase (S-adenosyl-L-methionine methylthioadenosine-lyase EC4.4.1.14) (ACC
synthase) (Yu et al., 1979). Genomic studies in many plant species has revealed a
divergent multigene family encoding this protein (Botella et al., 1993; Oetiker et al.,
1997; Kathiresan et al., 1998; Gonzalez and Botella, 2003; Yamagami et al., 2003). The
majority of the family members have specific expression patterns, which probably help to
adjust plant responses to particular environmental changes.
Three genes for ACC synthase have been cloned so far in pineapples and two of
them characterized in our laboratory (Cazzonelli et al., 1998; Botella et al., 2000). The
genes, ACACS1 and ACACS2 were amplified with ACC synthase degenerate oligo-
nucleotides from reverse transcribed total RNA extracted from ripening fruit or from low
temperature stressed leaf tissue, respectively. The ACACS1 was shown to be expressed in
fruits and in wounded leaves (Cazzonelli et al., 1998) while ACACS2 expression is
proposed to be associated with flowering (Botella et al., 2000).
In this study we describe a four year field trial and comparative analysis of the
first transgenic pineapple plants. We demonstrate that constitutive over-expression of an
ACACS2 gene fragment caused methylation of the endogenous ACACS2 gene resulting in
expression suppression in a considerable part of transgenic plants. Continuous monitoring
of the flowering dynamics of transgenic and control plants during two successive
generations displayed that suppression of the ACACS2 gene resulted in significantly
delayed flowering.
MATERIALS AND METHODS

Description of Transgenic Plants

Two independent transgenic lines of Smooth Cayenne pineapple were obtained
from DNA Plant Technology Corporation (USA) as embriogenic tissue transformants.
The inserted DNA consists of the following elements. The left and right border regions of
T-DNA from an octopine strain of A. tumefaciens (Gielen et al., 1984; Vandenelzen et al.,
1985; Komari et al., 1986). An enhanced 35S promoter from the cauliflower mosaic virus
(Kay et al., 1987) was used in combination with the waxy leader, derived from Zea mays
(Klosgen et al., 1986). In order to enhance gene expression in pineapple, an intron derived
from the CHS-A gene of Petunia hybrida (Koes et al., 1989) was inserted into the central
region of the waxy leader. This promoter-leader-intron structure was linked to the 5 end
of a 0.97 kbp fragment of an incomplete cDNA copy of the ACACS2 message (Botella et
al., 2000). The octopine synthase 3 region (terminator sequence) (Macdonald et al., 1991)
was linked to the end of the AC-ACS 2 fragment. The selection cassette contains a
tobacco acetolactate synthase gene (surB) (Lee et al., 1988) linked to the Ubi I promoter
from maize (Zea mays) (Christensen et al., 1992) at the 5 end and its own 3 UTR, to
serve as a selectable marker in plants cells.
Presence of the inserted T-DNA has been confirmed by PCR analysis of individual
plants using internal primers for the surB gene with expected fragment size of 449 bp.
Southern blot hybridization with 0.97 kb ACACS2 gene fragment as a probe against

30
genomic DNA extracted from a control Smooth Cayenne pineapple plant and plants
representing both transgenic lines revealed a single copy of inserted ACACS2 fragment in
each line.
Chemicals
Ethephon solution was prepared from 1:2000 dilutions of commercial Ethrel (May
and Baker, Australia). Auxin induction solution: 50 mg of sodium -naphthalene acetate
(NAA) (Sigma) were dissolved in 1L of distilled water.
Molecular Analysis Procedures
Pineapple RNA and DNA for blot hybridization were extracted as described
previously (Cazzonelli et al., 1998). Southern blot was performed on genomic DNA
digested with HindIII enzyme and hybridized with ACACS2 gene fragment. DNA for
PCR was extracted from approximately 100 mg of ground leaf tissue in extraction buffer
(150 mM Tris base, 2% (w/v) SDS, 50 mM EDTA, 1% -mercaptoethanol, adjusted to pH
7.5 with boric acid) and purified using GeneClean DNA isolation kit and optimised
protocol. 100 l of the centrifuged at 13000 rpm for 5 min extraction buffer containing
DNA was added 300 l of NaI solution and 5 l of glassmilk. After 3 min incubation on
bench the mixture was centrifuged at 13000 rpm for 1 min and supernatant was discarded.
The pellet washed 3 times with 500 l of the kit wash solution and vacuum dried for 15
min. DNA was eluted with 20 l of sterile water.

RESULTS AND DISCUSSION

Somaclonal Variation in Transgenic and Control Plants
Pineapple tissue culture propagation with the use of plant hormones unavoidably
causes alteration in gene expression and mutations, which result in elevated levels of
somaclonal variation (Firoozabady and Moy, 2004). Since cultivated pineapples are
propagated vegetatively, somaclonal variation could be inherited and maintained through
generations. Once the first generation of the transgenic and control tissue culture (TC)
propagated plants was established in the field, morphological analysis revealed substantial
levels of abnormalities in comparison with common field grown pineapples of the same
Smooth Cayenne cultivar.
To minimize the genetic variability, which could conceal or diminish the effects
caused explicitly by the transgene, not by random mutations, we used TC plants as a
control. These control plants were recovered from non-transformed cells, which
originated from the same callus and went through all transformation procedures as the
transformed cells, however, did not carry the transgene. Thus, each transgenic line has its
own TC control plants.
The most abundant aberration was an appearance of spikes on leaf edges. In total
59.5% of the plants developed this trait distributed as shown in Table 1. The differences
between transformed and TC control plants were found to be not statistically significant.
Other abnormalities such as lack of antocyanin production, stripes on leaves, intense
green leaves, inability to develop fruit despite of flowering and fruits without crown were
rare and distributed randomly across transgenic and TC control plants (Table 1).
Smooth Cayenne pineapples probably are a heterozygous variety with many loci
represented by alternative alleles (Collins and Kerns, 1938). The spikiness of leaves is
considered to be a genetically unstable character (Collins and Kerns, 1938), which is
under control of two genes with complex interrelationships (Collins and Kerns, 1946;
Usberti et al., 1995). Thus, such high level of spiny plants after tissue culture propagation
probably should be ascribed to an increased instability of the trait rather than direct
mutations as in case of the other defects. However, despite the instability the spines were
inherited during two successive generations in all those plants.
Since the tissue culture propagation affects the entire genome (in case of both
genetically stable and unstable genes) we also used specific control plants for each
transgenic line in further studies.

31
Comparative Analysis of Flowering Dynamics
Once isolated from cold stressed pineapple leaves ACACS2 gene was also shown
to be upregulated by an auxin treatment (Botella et al., 2000). In case of pineapple the
inducibility with low temperature and auxin is a good indication that this gene might be
responsible for the ethylene burst responsible for initiating flowering.
To assess the ability of ACACS2 to control flowering in pineapple we obtained two
independent transgenic lines over-expressing a truncated copy of the gene, originated
from an incomplete 0.97 kb cDNA fragment. The plants were established in the field and
allowed to flower naturally. Monitoring of inflorescence appearance in each transgenic
line and corresponding control plants was performed during six months (from September
2001 till February 2002). Dynamics of natural flowering over this period clearly
demonstrated that both transgenic lines had lower flowering activity. Using the chi-square
method we have shown that decreased flowering rates of the transgenic plants in both
lines were highly statistically significant for one degree of freedom at the 0.001% level of
significance.
To test the ability of the plants to produce flowers in response to artificial agents
the rest of the plants were treated with ethephon, a chemical commonly used by pineapple
growers for flowering induction. The flowering dynamics was monitored in the same way
as previously. Majority of the plants developed flowers within the first two months after
the treatment. The rates of flowering of transgenic and control plants did not display any
significant difference.
The reduced flowering in transgenic plants before ethephon treatment and similar
flowering rates in transgenic and control plants after the treatment concurs with our
original idea. In fact, the transgenic plants are expected to be impaired in endogenous
ethylene production, which assumingly should result in flowering delay or obliteration. At
the same time treatment with ethephon releasing ethylene should compensate the lack of
endogenously produced ethylene and trigger the flowering similarly in transgenic and
control plants.
For the next generation, similar size slips were selected and established in a neigh-
boring field on March 2003. During January-October 2004 the plants were assessed for
flowering in the same way as described above and transgenic plants for a second time
demonstrated similar reduction of the flowering capacity. We also analyzed the
inheritance of the suppressed flowering. The progeny of transgenic plants that had
previously displayed delayed flowering and transgenic plants that had flowered normally
were compared and found to be significantly different. The former group started to flower
considerably later than the second group that surprisingly demonstrated flowering rates
very similar to that of control plants (progeny of the TC control plants) (Fig. 1).
The observed difference among transgenic plants was not restricted to one line and
was similarly represented in progenies of line 1 and line 2. The fact that a fraction of
transgenic plants exhibited normal flowering behavior in the first generation suggests that
the mere presence of the transgene was not sufficient to cause the flowering delay. To
solve this paradox we assumed that the over-expression of the ACACS2 fragment resulted
in co-suppression of the endogenous ACACS2 gene only in a part of transgenic plants,
while the other part has this gene functionally intact. This possibility does not by any
means discard data obtained during the first generation, however, the second generation
data allows estimation of the flowering delay caused by suppression of the ACACS2 gene.
Methylation Is a Possible Mechanism for Co-Suppression of the ACACS2 Gene
To explain the fact that delayed flowering was not displayed by all transgenic
plants, we attempted to clarify the mechanism of co-suppression. Methylation has been
suggested as a general mechanism for gene silencing and co-suppression associated with
transposon inactivation, transformation with foreign DNA, expression of aberrant RNA
and RNA interference (Russo et al., 1996; Curradi et al., 2002; Jaenisch and Bird, 2003).
After analysis of flowering delay in the second generation we selected transgenic plants
with consistently delayed and normal flowering. To test whether the methylation occurred

32
in transgenic plants we subjected genomic DNA extracted from these plants to digestion
with methylation sensitive enzyme BstUI. Southern blot hybridization with ACACS2
cDNA as a probe revealed that patterns of bands were drastically different in those
transgenic plants that exhibited different flowering behaviour. The difference was in
agreement with the distribution of CGCG restriction sites in the inserted T-DNA sequence
and indicated the occurrence of methylation.
To confirm that the methylation resulted in downregulation of the gene expression
we performed Northern blot analysis of total RNA, extracted from transgenic plants
possessing methylated and not methylated ACACS2 genes and from non transgenic
control plants after induction with auxin (50 mg/L NAA). ACACS2 gene was induced in
control and transgenic plant with the non-methylated ACACS2 gene, while there was no
expression detected in transgenic plants with the methylated ACACS2 gene.

CONCLUSION
This work in a certain respect culminates a long succession of previous studies by
different authors, which have provided valuable information about flowering control in
pineapple and discovered a plethora of intermediate factors leading to the activation of a
particular gene. This gene, ACACS2 encoding an ACC synthase enzyme activated
specifically prior flowering, seems to be one of the keys switching meristematic cells
from vegetative to generative development by means of the plant hormone ethylene. We
have shown that suppression of ACACS2 transcription in transgenic pineapple plants
resulted in significant flowering delay, however, did not prevent it completely. Our data
demonstrated that ACACS2 gene is a potent regulator of flowering in pineapples and
suppression of its activity could be successfully used to control natural flowering in
commercial situations.
ACKNOWLEDGEMENTS
This research was funded, in part, by grants from the Australian Research Council,
and by funds from Golden Circle Ltd. (Australia).
Literature Cited
Adams, D.O. and Yang, S.F. 1979. Ethylene biosynthesis - identification of 1-
Aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of
methionine to ethylene. Proc. Natl. Academy of Sciences of the USA 76:170-174.
Bartholomew, D.P. 1977. Inflorescence development of pineapple (Ananas comosus L.
Merr.) induced to flower with ethephon. Botanical Gazette 138:312-320.
Bartholomew, D.P. 1987. Effects of environment on pineapple growth and development.
HortScience 22:1079-1079.
Botella, J.R., Cavallaro, A.S. and Cazzonelli, C.I. 2000. Towards the production of
transgenic pineapple to control flowering and ripening. Acta Hort. 529:115-122.
Botella, J.R., Schlagnhaufer, C.D., Arteca, J.M., Arteca, R.N. and Phillips, A.T. 1993.
Identification of two new members of the 1-Aminocyclopropane-1-carboxylate
synthase encoding multigene family in mung bean. Gene 123:249-253.
Burg, S.P. and Burg, E.A. 1966. Auxin-induced ethylene formation: its relation to
flowering in pineapple. Science 152:1269.
Cazzonelli, C.I., Cavallaro, A.S. and Botella, J.R. 1998. Cloning and characterisation of
ripening-induced ethylene biosynthetic genes from non-climacteric pineapple (Ananas
comosus) fruits. Australian J. Plant Physiol. 25:513-518.
Christensen, A.H., Sharrock, R.A. and Quail, P.H. 1992. Maize polyubiquitin genes -
structure, thermal perturbation of expression and transcript splicing, and promoter
activity following transfer to protoplasts by electroporation. Plant Molec. Biol.
18:675-689.
Collins, J.L. and Kerns, K.R. 1938. Mutations in the pineapple. J. Heredity 29:162-172.
Collins, J.L. and Kerns, K.R. 1946. Inheritance of three leaf types in the pineapple. J.
Heredity 37:123-128.

33
Curradi, M., Izzo, A., Badaracco, G. and Landsberger, N. 2002. Molecular mechanisms of
gene silencing mediated by DNA methylation. Molec. Cell. Biol. 22:3157-3173.
Firoozabady, E. and Moy, Y. 2004. Regeneration of pineapple plants via somatic
embryogenesis and organogenesis. In Vitro Cellular & Developmental Biology-Plant
40:67-74.
Friend, D.J.C. and Lydon, J. 1979. Effects of daylength on flowering, growth, and cam of
pineapple (Ananas comosus L. Merrill). Botanical Gazette 140:280-283.
Gielen, J., Debeuckeleer, M., Seurinck, J., Deboeck, F., Degreve, H., Lemmers, M.,
Vanmontagu, M. and Schell, J. 1984. The complete nucleotide sequence of the t-DNA
of the Agrobacterium tumefaciens plasmid Ptiach5. Embo J. 3:835-846.
Gonzalez, N. and Botella, J.R. 2003. Characterisation of three ACC synthase gene family
members during post-harvest-induced senescence in broccoli (Brassica oleracea L.
var. italica). J. Plant Biol. 46:223-230.
Jaenisch, R. and Bird, A. 2003. Epigenetic regulation of gene expression: how the
genome integrates intrinsic and environmental signals. Nature Genetics 33:245-254.
Kathiresan, A., Nagarathna, K.C., Moloney, M.M., Reid, D.M. and Chinnappa, C.C.
1998. Differential regulation of 1-aminocyclopropane-1-carboxylate synthase gene
family and its role in phenotypic plasticity in Stellaria longipes. Plant Molec. Biol.
36:265-274.
Kay, R., Chan, A., Daly, M. and McPherson, J. 1987. Duplication of Camv-35s promoter
sequences creates a strong enhancer for plant genes. Science 236:1299-1302.
Klosgen, R.B., Gierl, A., Schwarzsommer, Z. and Saedler, H. 1986. Molecular analysis of
the waxy locus of Zea mays. Molec. General Genet. 203:237-244.
Koes, R.E., Spelt, C.E., Vandenelzen, P.J.M. and Mol, J.N.M. 1989. Cloning and
molecular characterization of the chalcone synthase multigene family of Petunia
hybrida. Gene 81:245-257.
Komari, T., Halperin, W. and Nester, E.W. 1986. Physical and functional map of super-
virulent Agrobacterium tumefaciens tumor-inducing plasmid Ptibo542. J. Bacteriol.
166:88-94.
Kuan, C.S., Yu, C.W., Lin, M.L., Hsu, H.T., Bartholomew, D.P. and Lin, C.H. 2005.
Foliar application of aviglycine reduces natural flowering in pineapple. HortScience
40:123-126.
Lee, K.Y., Townsend, J., Tepperman, J., Black, M., Chui, C.F., Mazur, B., Dunsmuir, P.
and Bedbrook, J. 1988. The molecular basis of sulfonylurea herbicide resistance in
tobacco. Embo J. 7:1241-1248.
Macdonald, M.H., Mogen, B.D. and Hunt, A.G. 1991. Characterization of the
polyadenylation signal from the t-DNA-encoded octopine synthase gene. Nucleic
Acids Research 19:5575-5581.
Manica, I., Fioravanco, J.C., Barradas, C.I.N., Kist, H. and Vione, G.F. 1994. Flowering
induction and yield of pineapple cv. Smooth Cayenne. Pesquisa Agropecuaria
Brasileira 29:81-86.
Min, X.J. and Bartholomew, D.P. 1996. Effect of plant growth regulators on ethylene
production, 1-aminocyclopropane-1-carboxylic acid oxidase activity, and initiation of
inflorescence development of pineapple. J. Plant Growth Regul. 15:121-128.
Oetiker, J.H., Olson, D.C., Shiu, O.Y. and Yang, S.F. 1997. Differential induction of seven
1-aminocyclopropane-1-carboxylate synthase genes by elicitor in suspension cultures
of tomato (Lycopersicon esculentum). Plant Molec. Biol. 34:275-286.
Randhawa, G.S., Dass, H.C. and Chacko, E.K. 1970. Effect of ethrel, NAA and NAD on
induction of flowering in pineapple (Ananas comosus L.). Curr. Sci. 39:530-&.
Reid, M.S. and Wu, M.J. 1991. Ethylene in flower development and senescence. p.215-
234. In: A.K. Mattoo and J.C. Suttle (eds.), The plant hormone ethylene, CRC Press
Inc., Boca Raton, Florida.
Reinhardt, D. and Medina, V.M. 1992. Growth and fruit quality of Perola and Smooth
Cayenne pineapple cultivars. Pesquisa Agropecuaria Brasileira 27:435-447.
Reinhardt, D., Costa, J.T.A. and Dacunha, G.A.P. 1986. Influence of planting date, of

34
 rejection rate, and of age of the plant for floral inducement of the smooth cayenne
pineapple in reconcavo-baiano (Brazil). 1. Vegetative growth, production of rejection
and natural flowering. Fruits 41:31-41.
Russo, X., Martienssen, R.A. and Riggs, A.D. 1996. Epigenetic mechanisms of gene
regulation. Plainview, NY.: Cold Spring Harbor Laboratory Press.
Usberti, J.A., Siqueira, W.J., Spironello, A., Harris, M. and Badan, A.C.D. 1995.
Inheritance of leaf spininess and segregation of leaf color in pineapple (Ananas
comosus L. Merrill). Brazilian J. Genetics 18:547-552.
Vandenelzen, P., Lee, K.Y., Townsend, J. and Bedbrook, J. 1985. Simple binary vectors
for DNA transfer to plant cells. Plant Molec. Biol. 5:149-154.
Vieira, A., Gadelha, R.S.D., Maldonado, J.F.M. and Dossantos, A.C. 1983. Plant age and
its influence in the flowering induction and yield of pineapple cultivar Smooth
Cayenne. Pesquisa Agropecuaria Brasileira 18:33-35.
Yamagami, T., Tsuchisaka, A., Yamada, K., Haddon, W.F., Harden, L.A. and Theologis, A.
2003. Biochemical diversity among the 1-amino-cyclopropane-1-carboxylate synthase
isozymes encoded by the Arabidopsis gene family. J. Biol. Chem. 278:49102-49112.
Yu, Y.B., Adams, D.O. and Yang, S.F. 1979. 1-Aminocyclopropanecarboxylate synthase, a
key enzyme in ethylene biosynthesis. Archives of Biochemistry and Biophysics
198:280-286.

35
Tables

Table 1. Somaclonal variation.

Line 1 TC control 1 Line 2 TC control 2 Field grown

Total number of plants 111 328 108 83 100
Percentage of spiny plants 54.9 70.1 46.3 40.9 0
Number of plants 4 1 1 0 0
without antocyanin
Number of plants 0 0 1 0 0
without crown
Number of plants with 0 0 1 2 0
crest crown
Number of plants with 2 1 0 0 0
stripes on leaves
Number of plants with 2 0 1 0 0
intense green leaves
Number of plants 2 11 2 5 0
without fruits

Figurese

60

progeny of tranformants with delayed flowering

Percentage of flowered plants

50
progeny of tranformants with normal flowering
non transgenic control
40

30

20

10

0
Oct-03 Nov-03 Dec-03 Jan-04 Feb-04 Mar-04 Apr-04 May-04 Jun-04 Jul-04 Aug-04 Sep-04 Oct-04 Nov-04

Fig. 1. Flowering dynamics of progeny of the transgenic and control plants.

36