JXB Advance Access originally published online on October 17, 2006
Journal of Experimental Botany 2006 57(14):3953-3960; doi:10.1093/jxb/erl167
RESEARCH PAPER |
Silencing of the ACC synthase gene ACACS2 causes delayed flowering in pineapple [Ananas comosus (L.) Merr.]
Plant Genetic Engineering Laboratory, Department of Botany, School of Integrative Biology, University of Queensland, Brisbane 4072, Australia
* To whom correspondence should be addressed. E-mail: j.botella{at}uq.edu.au
Received 20 June 2006; Accepted 22 August 2006
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Abstract |
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Flowering is a crucial developmental stage in the plant life cycle. A number of different factors, from environmental to chemical, can trigger flowering. In pineapple, and other bromeliads, it has been proposed that flowering is triggered by a small burst of ethylene production in the meristem in response to environmental cues. A 1-amino-cyclopropane-1-carboxylate synthase (ACC synthase) gene has been cloned from pineapple (ACACS2), which is induced in the meristem under the same environmental conditions that induce flowering. Two transgenic pineapple lines have been produced containing co-suppression constructs designed to down-regulate the expression of the ACACS2 gene. Northern analysis revealed that the ACACS2 gene was silenced in a number of transgenic plants in both lines. Southern hybridization revealed clear differences in the methylation status of silenced versus non-silenced plants by the inability of a methylation-sensitive enzyme to digest within the ACACS2 DNA extracted from silenced plants, indicating that methylation is the cause of the observed co-suppression of the ACACS2 gene. Flowering characteristics of the transgenic plants were studied under field conditions in South East Queensland, Australia. Flowering dynamics studies revealed significant differences in flowering behaviour, with transgenic plants exhibiting silencing showing a marked delay in flowering when compared with non-silenced transgenic plants and control non-transformed plants. It is argued that the ACACS2 gene is one of the key contributors towards triggering ‘natural flowering’ in mature pineapples under commercial field conditions.
Key words: ACC synthase, co-suppression, ethylene, pineapple flowering, transgenic pineapple
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Flowering is one of the most important processes in plant ontogeny, consisting of the transition from vegetative growth to generative development that ultimately allows reproduction. Onset of flowering in pineapples is marked by the appearance of a small red inflorescence in the centre 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, it can be triggered in immature plants by environmental stresses (Friend and Lydon, 1979; Reinhardt et al., 1986; Bartholomew, 1987; Min and Bartholomew, 1996). Synchronizing the flowering of plants in the field has a critical importance for the pineapple industry 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 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 fraction of the crop (ranging from 5% to 30% and reaching up to 70% under certain conditions) manages to flower ahead of schedule, a phenomenon known as ‘natural flowering’ or ‘environmental induction’ (Min and Bartholomew, 1996). This is a highly undesirable characteristic of pineapples grown worldwide, causing disruption in harvest scheduling and market supply, increasing harvest costs (multiple harvests of the same field), and resulting in significant harvest loses (Min and Bartholomew, 1996). Given the difficulty of synchronizing flowering by chemical or agronomical means, a possible solution to this problem is to postpone or prevent ‘natural flowering’ in field-grown pineapple crops using genetic engineering techniques.
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 such as ethephon [(2-chloroethyl)phosphonic acid] 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 stimulation of ethylene production (Burg and Burg, 1966). Despite the existence of ethylene biosynthesis inhibitors and chemicals disrupting ethylene signalling, attempts to arrest or at least delay unwanted flowering in pineapple with these agents have had little success (Min and Bartholomew, 1996; Kuan et al., 2005).
The key regulatory enzyme in the ethylene biosynthetic pathway is 1-amino-cyclopropane-1-carboxylate synthase (S-adenosyl-L-methionine methylthioadenosine-lyase EC 4.4.1.14 [EC] ) (ACC synthase) (Yu et al., 1979). Three genes for ACC synthase have been cloned so far in pineapples and two of them have been characterized (Cazzonelli et al., 1998; Botella et al., 2000). The genes, ACACS1 and ACACS2, were amplified by polymerase chain reaction (PCR) using degenerate oligonucleotides from reverse-transcribed total RNA extracted from ripening fruit or from low temperature-stressed leaf tissue, respectively. 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, the results of a field trial and the comparative analysis of transgenic pineapple plants containing co-suppression constructs designed to inhibit ethylene biosynthesis under natural flowering-inducing conditions are described. It is shown that constitutive overexpression of an ACACS2 gene fragment causes methylation of the endogenous ACACS2 gene resulting in silencing. Continuous monitoring of the flowering dynamics of transgenic and control plants showed that suppression of the ACACS2 gene resulted in significantly delayed flowering.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Production of transgenic plants
Two independent transgenic lines of ‘Smooth Cayenne’ pineapple were obtained from the DNA Plant Technology Corporation (USA). The production of the transgenic pineapples by Agrobacterium-mediated transformation has been described elsewere (Firoozabady and Gutterson, 1998; Firoozabady and Moy, 2004; Firoozabady et al., 2006). The inserted T-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 maize (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 kb 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 ACACS2 fragment. The selection cassette contains a tobacco acetolactate synthase gene (surB) (Lee et al., 1988) linked to the Ubi I promoter from maize (Christensen et al., 1992) at the 5' end and its own 3'-untranslated region (UTR), to serve as a selectable marker in plant cells.
The presence of the inserted T-DNA was confirmed by PCR analysis of individual plants using internal primers for the surB gene with an expected fragment size of 449 bp.
Plants grown in tissue culture and subjected to transformation procedures but not containing the transgenic construct were used as control and named TC.
Field trial
Plants were grown in a randomized plot at the Department of Primary Industries ‘Redlands Research Station’ in the Redlands Shire, Brisbane, Australia. Planting of the T0 generation took place in mid-April, 2000. The following generation was planted at the end of March, 2003. In both cases, plants were planted in double-row beds at 1.82 m centres (oriented north–south). The distance between the plants within the rows was 
33 cm (which roughly corresponds to 40 000 plants ha–1).
Chemicals
The calculated amount of ethephon solution was prepared from 1:2000 dilutions of commercial Ethrel (May and Baker, Australia) and was sprayed to the point of runoff with a final application volume of 30 ml plant–1. Auxin induction solution: 50 mg of sodium 
-naphthalene acetate (NAA) (Sigma) were dissolved in 1.0 l of distilled water; 10 ml of the solution was poured down the heart of each plant tested.
Molecular analysis procedures
Pineapple genomic DNA was extracted as previously described (Cazzonelli et al., 1998). Southern blot was performed on genomic DNA digested with HindIII or BstUI enzymes, and hybridized with the 0.97 kb ACACS2 gene fragment. DNA for PCR analysis was extracted from 100 mg of ground leaf tissue in extraction buffer [150 mM TRIS base, 2% (w/v) SDS, 50 mM EDTA, 1% ß-mercaptoethanol, adjusted to pH 7.5 with boric acid] and purified using a GeneClean DNA isolation kit (Q-BIOgene). RNA was extracted from leaf bases of mature plants treated with NAA (50 mg l–1) or water as previously described (Cazzonelli et al., 1998). Northern blot hybridization was performed as described by Cazzonelli et al. (1998).
Statistical analysis
The alterations in phenotype and flowering dynamics in transgenic versus control groups were tested for statistical significance by the 
2 method. The differences were considered significant if a 
95% level of probability was reached.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Somaclonal variation in transgenic and control plants
Two independent transgenic lines were obtained using a genetic construct containing an incomplete 0.97 kb cDNA fragment of the ACACS2 gene under the control of an enhanced cauliflower mosaic virus 35S promoter (Kay et al., 1987). After transformation, clonal propagation in tissue culture was used to produce a total of 111 plants for line 1 and 108 for line 2. The presence of the transgene was verified by PCR analysis in all individual plants present in the field trial and by Southern blot hybridization of selected plants from both lines (Fig. 1A, B). To minimize the genetic variability, which could conceal or diminish the effects explicitly caused by the transgene, and not by random mutations, two types of control plants were also present in the field trial. The first one, named TC control plants in this work, consisted of pineapple plants that had undergone transformation and regeneration processes similar to the transgenic plants but were shown to be non-transgenic by PCR screening, (commonly known as escapes). Each of the two transgenic lines was generated in independent transformation experiments; therefore, for each transgenic line, the corresponding TC control plants were recovered from non-transformed cells originating from the same calli. Thus, each transgenic line had its own TC control plants. The second kind of controls consisted of normal pineapple plants that had not undergone any tissue culture. Plants were grown in a totally randomized plot for analysis.
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Morphological alterations as a result of somaclonal variation are a usual phenomenon observed in plants regenerated from tissue culture (Hossain et al., 2003; Charlton et al., 2004; Ravindra et al., 2004). In pineapple, the use of plant hormones for tissue culture unavoidably causes alterations 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 can 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 (Table 1).
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The most abundant aberration observed in the field was the appearance of spines on leaf edges. The differences between transformed and TC control plants were not statistically significant. Other abnormalities were rare and distributed randomly across transgenic and TC control plants (Table 1).
Smooth Cayenne pineapples are believed to be a heterozygous variety with many loci represented by alternative alleles (Collins and Kerns, 1938). Leaf spininess is considered to be an unstable character, which is under the control of a single genetic locus with three possible alleles (Kinjo, 1993; Cabral et al., 1997; Carlier et al., 2004; Kato et al., 2004). Thus, the high percentage of spiny plants observed after tissue culture propagation is most probably due to an increased instability in these particular genes rather than direct mutations as in the case of the other defects.
A percentage of transgenic plants show silencing of the ACACS2 gene
Analysis of flowering dynamics was performed in the first generation of transgenic plants grown directly from tissue culture, revealing that, in general, transgenic plants displayed flowering behaviour similar to the control plants (Fig. 1C). Statistical analysis, however, showed that transgenic plants in both lines had a lower number of flowering plants over the first 6 month period after planting (significant for one degree of freedom at the <0.001% level of significance) (Table 2).
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A second field trial was performed using vegetatively propagated progeny of the plants used in the first trial. Once these plants were established in the field, ACACS2 expression levels were studied in the offspring of early and late flowering transgenic plants as well as TC control plants (Fig. 2A). In control plants, ACACS2 basal levels are low but clearly detectable; in addition, auxin treatment (50 mg l–1 NAA) resulted in strong induction of the gene. The early flowering transgenic plants show high basal levels of ACACS2 signal, probably due to the constitutive expression of the inserted ACACS2 fragment. Auxin treatment of these plants results in even higher levels due to the enhanced expression of the endogenous gene in addition to the ACACS2 RNA pool produced from the inserted transgene (Fig. 2A). It is noteworthy that the size of the mRNA resulting from the inserted genetic construct is predicted to be very similar to the native ACACS2 mRNA, therefore it has a similar position on the blot. In the late flowering transgenic plants, however, ACACS2 levels are almost undetectable and auxin induction fails to produce any increase in transcript levels, indicating that both the native ACACS2 gene as well as the inserted transgene have been silenced.
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Methylation is a possible mechanism for co-suppression of the ACACS2 gene
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). To test the occurrence of methylation, genomic DNA extracted from early and late flowering transgenic plants as well as control plants was digested with the methylation-sensitive enzyme BstUI. Figure 2B shows the results of Southern blot hybridization experiments using the ACACS2 cDNA as a probe. The banding patterns observed in control plants are in a good agreement with the occurrence of CGCG tetranucleotides (BstUI recognition site) within the ACACS2 sequence. The four bands observed in the control lane suggest the presence of at least three restrictions sites within the native ACACS2 gene. In fact, the ACACS2 cDNA contains four CGCG sites, generating theoretical internal fragments of 252, 137, and 18 bp, plus two additional fragments containing the outward fragments of the gene (Fig. 2C).
Digestion of genomic DNA from transgenic plants with BstUI should theoretically produce the same fragments observed in control plants plus two extra fragments of 199 and 920 bp originating from the inserted T-DNA. Indeed, Southern blot of early flowering transgenic plants shows the expected theoretical pattern of bands (Fig. 2B). These plants did not show any silencing of the ACACS2 gene (Fig. 2A). In contrast, the lane corresponding to the late flowering transgenic plants (showing silencing of the transgene and native ACACS2 gene) shows a dramatically different banding pattern. The 199 and 920 bp fragments observed in the early flowered transgenics are not present and, most importantly, the low molecular weight bands corresponding to the endogenous ACACS2 gene have also disappeared, strongly suggesting the occurrence of methylation in both the transgene and the endogenous gene.
Comparative analysis of flowering dynamics
Unlike many other plants, pineapples can be forced to flower by exogenous application of the gaseous hormone ethylene. This observation resulted in the hypothesis that an ethylene-based flowering initiating mechanism could exist in pineapples (Burg and Burg, 1966; Randhawa et al., 1970). The pathway for ethylene synthesis in plants is well established (Adams and Yang, 1979) and it has been shown that ACC synthase is a key enzyme in the pathway (Yu et al., 1979). Studies in several plant species have revealed that this protein is encoded by a divergent multigene family (Botella et al., 1993; Oetiker et al., 1997; Kathiresan et al., 1998; Gonzalez and Botella, 2003; Yamagami et al., 2003). The members of the family exhibit different tissue-specific expression, different induction patterns by hormones and environmental stimuli, and the proteins have different biochemical properties and catalytic activities (Yamagami et al., 2003).
Transgenic and control plants from both lines were established in the field and grown to maturity under normal conditions. Since only a fraction of the transgenic plants displayed silencing of the ACACS2 gene, flowering dynamics were monitored separately for silenced and non-silenced transgenics and TC control plants. Each group of plants was represented by at least 100 plants.
Monitoring was performed weekly between October 2003 and November 2004, the flowering date recorded for each plant, and the percentages of plants with newly appeared inflorescences calculated for each group of plants each month. Figure 3 shows the distribution of flowering events for the silenced and non-silenced transgenics as well as TC controls in each of the two lines. All plants showed a similar total flowering capacity; however, transgenic plants with the silenced ACACS2 gene started to flower later than control or non-silenced plants. The average number of days from planting until appearance of the inflorescence for each group in each of the two lines was estimated with an accuracy of 1 week (Fig. 4). The delay calculated as the difference between the average flowering dates for the silenced transgenic plants versus TC controls was 69.4 and 52.1 d for line 1 and line 2, respectively, and was statistically significant (P <0.05) (Fig. 4).
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The ACACS2 cDNA was isolated from leaves of pineapple plants subjected to cold stress and was also shown to be up-regulated by an auxin treatment (Botella et al., 2000). This dual inducibility suggests that ACACS2 might be responsible for triggering flowering in pineapples by elevating the ethylene level in the competent meristem, since both factors (low temperature and auxin) are known to induce flowering in pineapple (Burg and Burg, 1966; Bartholomew, 1987). The results show that silencing of the ACACS2 gene significantly delays flowering in pineapples. However, since the silencing was achieved by co-suppression, there is a possibility that more than one member of the ACC synthase family was silenced. Arguing against this possibility is the fact that co-suppression requires substantial sequence identity (Depicker and VanMontagu, 1997; Kunz et al., 2001) and ACC synthase genes share very little conservation. In fact, sequence analysis of all ACC synthase sequences known in pineapple revealed significant diversity among them, with the highest level of identity being only 59% between ACACS1 and ACACS2. In addition, Southern blot analysis failed to reveal any cross-hybridization of ACACS2 with any other ACC synthase genes (Botella et al., 2000), thus providing further evidence supporting the divergence of the ACC synthase gene family in pineapples. Therefore, the observed delay is probably caused by the silencing of the ACACS2 gene.
If inactivation of the ACACS2 gene causes a reduction in ethylene synthesis in the environmental conditions necessary for flowering induction, exogenously applied ethylene should restore the flowering in the silenced plants. To test the ability of the plants to flower in response to artificial agents, 20 plants of each group were treated with ethephon and the flowering monitored as described above. The majority of the plants developed flowers within the first 2 months after the treatment, and flowering rates in silenced and non-silenced transgenic as well as in control plants did not display any significant differences (data not shown), further suggesting that insufficient synthesis of ethylene was the cause of the observed flowering delay in transgenic plants with the silenced ACACS2 gene.
In conclusion, the ACC synthase gene ACACS2 could be a key element in the production of the ethylene burst that switches meristematic cells from vegetative to generative development in pineapple. It has been shown that silencing of ACACS2 in transgenic pineapple plants results in a significant flowering delay; however, it does not prevent it indefinitely. The data strongly suggest that ACACS2 can be the trigger of the ‘natural flowering’ phenomenon observed in pineapple plantations under field conditions. The results also prove that silencing of the ACACS2 gene using genetic engineering techniques can be successfully used to control natural flowering in commercial situations, therefore addressing this major pineapple industry problem.
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Acknowledgements |
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This research was funded by a grant from the Australian Research Council LP0980025164, and Golden Circle Ltd (Australia).
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Abbreviations |
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ACC synthase, 1-amino-cyclopropane-1-carboxylate synthase; NAA,
-naphthalene acetate; PCR, polymerase chain reaction. |
References |
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