The role of PnACO1 in light- and IAA-regulated flower inhibition in Pharbitis nil
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In this study, the first ACC oxidase (PnACO1) cDNA from model short-day plant Pharbitis nil was isolated. The expression pattern of PnACO1 was studied under different conditions (photoperiod and auxin), an adequate balance of which determines P. nil flowering. It was shown that the gene was transcribed in all the examined organs of the 5-day-old seedling and was strongly activated by auxin. Our results also revealed that PnACO1 transcript accumulation in the cotyledons showed diurnal oscillations under both LD and SD conditions. On the basis of presented and previously obtained data, we suggest that flowering inhibition evoked by IAA in P. nil results from its stimulatory effect on both ACC synthase and oxidase gene expression and, consequently, enhances ethylene production.
KeywordsACC oxidase Auxin Ethylene Flowering Pharbitis nil (Ipomoea nil)
Plant hormones are factors that integrate individual metabolic processes required for a functional transformation of the vegetative meristem into a generative one. A search for a substance responsible for flower induction caused by a relevant photoperiod allowed many chemical compounds to be identified, which have either a stimulating or inhibiting effect on the flowering of the model short-day plant (SDP) Pharbitis nil. The former group consists of gibberellins (Galoch et al. 2002; King et al. 1987; Wijayanti et al. 1996), cytokinins (Friedman et al. 1990; Galoch et al. 1996; Halevy et al. 1991) and prostaglandins (Groenewald and van der Westhuizen 2001). The latter group consists of auxins (Kulikowska-Gulewska et al. 1995; Wijayanti et al. 1997), ethylene (Amagasa and Suge 1987; Kęsy et al. 2008), jasmonic acid (Maciejewska and Kopcewicz 2003), abscisic acid (Wijayanti et al. 1997; Wilmowicz et al. 2008) and brassinosteroids (Kęsy et al. 2003). Among flowering inhibitors, the strongest effect is demonstrated by auxins and ethylene. Most data used for determining their role in regulating this process in P. nil come mainly from typically physiological studies. However, there is little information on auxin and ethylene interactions at the molecular level. The identification of genes encoding for IAA and ethylene metabolism enzymes in P. nil, as well as the determination of changes in the level of their expression in cotyledons and shoot apices of plants cultivated under different photoperiodic conditions, will promote a better understanding of photoperiodic flower induction.
In P. nil, the highest auxin effectiveness was observed following its application to the cotyledons directly before or during the first half of the inductive night (Amagasa and Suge 1987; Friedman et al. 1990; Kulikowska-Gulewska et al. 1995; Ogawa and Zeevaart 1967). In turn, the level of endogenous auxin in the cotyledons of P. nil seedlings before and during the first half of the inductive darkness remained low, only growing between hours 8 and 12 into the night (Bodson 1985). Application of auxin together with AVG (an ethylene biosynthesis inhibitor) restored the inductive effect of the long night (Kęsy et al. 2008). Therefore, it seems that auxin’s inhibiting effect on P. nil flowering is achieved by an increased level of ethylene. It could be so, because it was proved that auxins induce the expression of some ACC synthases (Arteca and Arteca 1999; Frankowski et al. 2009; Kęsy et al. 2010; Tian et al. 2002; Tsuchisaka and Theologis 2004a), while the level of these genes’ expression correlates with the level of ethylene production (Abel et al. 1995; Kęsy et al. 2008; Overmyer et al. 2000; Smith and Arteca 2000; Tian et al. 2002; Tsuchisaka and Theologis 2004b).
Depending on the plant species and the environmental conditions, ethylene production can be controlled both at the level of gene expression and the ACS and ACO enzyme activity (Finlayson et al. 1999; Kathiresan et al. 1996, 1998; Machackova et al. 1997). Individual genes encoding for enzymes that participate in this hormone’s biosynthesis are expressed in diverse ways, with their products displaying different biochemical properties, which allows plants to respond to changing conditions in a time- and space-specific manner (Bleecker and Kende 2000; Kathiresan et al. 1996, 1998). Additionally, the accumulation level of transcripts of certain synthases (Frankowski et al. 2009; Kęsy et al. 2010), and perhaps also of ACC oxidases, is regulated by light and correlates with an increase in ethylene production (Kęsy et al. 2008).
In this study, the first ACO gene (designated PnACO1) from P. nil cotyledons was isolated and its expression levels were observed in the cotyledons of plants cultivated under both inductive and non-inductive photoperiodic conditions and following application of IAA. We also studied the PnACO1 expression pattern in various vegetative organs of plants cultivated under continuous light (non-inductive control) and both under long inductive night and IAA treatment.
Due to the great commercial significance of ethylene, which controls the processes of development, maturation and senescence of flowers and fruits, most of the experimental studies published deal only with the analyses of the changes in the expression levels and activity of ACC synthases and oxidases in those organs. Hence, the research results presented herein are of a pioneering nature.
Materials and methods
Seeds of Pharbitis nil Chois cv. Violet (Marutane Seed Co., Kyoto, Japan) were stirred with concentrated sulfuric acid for 45 min, washed under running tap water for 2 h and soaked for 24 h in water (26 ± 1 °C). The swollen seeds were sown in pots filled with vermiculite and sterile sand (1:1). The pots were transferred to a growth chamber at 26 ± 1 °C with continuous irradiance (130 μmol m−2 s−1 cool white fluorescent tubes Polam, Warsaw, Poland) for 5 days. IAA (0.5 mM) applications to both sides of the cotyledons were performed with the use of a small soft brush (about 50 μl per plant).
Plant material for PnACO1 cDNA cloning
IAA was applied to the cotyledons of 5-day-old plants. After 2 h of IAA application, the cotyledons (without petioles) were harvested, immediately frozen in liquid nitrogen and stored at −80 °C.
Expression analysis under photoperiodic conditions
Plants were grown under long-day (LD) conditions (16 h of light and 8 h of darkness) for 5 days. A portion of 5-day-old plants was left under these conditions (variant I). The remainder of the plants grew in 16-h-long darkness (variant II), and some of these were treated with IAA (variant III). Cotyledons (without petioles) were harvested every 2 h after IAA application or transfer to the darkness and, additionally, at hours 1, 9 and 17. Subsequently, the cotyledons were frozen in liquid nitrogen and stored at −80 °C.
Plants were grown under continuous light for 5 days and subsequently divided into four portions. The first was left under the same conditions; the second grew under continuous light, but IAA was applied at the beginning of the 6th day; the third was exposed to a 16-h-long inductive night; and the fourth was transferred to a 16-h-long night period and treated with IAA at the beginning of the 6th day. The apices, petioles, cotyledons, hypocotyls and roots were picked at hours 2, 4, 8 and 16 after the beginning of the 6th day, immediately frozen in liquid nitrogen and stored at −80 °C. All manipulations during the dark period were performed under dim green safe light. Each experiment was repeated at least three times (biological replicates). All data are presented as mean ± standard error (SE).
Molecular cloning of PnACO1
Pharbitis nil tissue (1.0–1.5 g) was homogenized in a sterile chilled mortar with a pestle. Total RNA was isolated with Tri Reagent (Sigma) and genomic DNA was removed with Deoxyribonuclease I (Fermentas). All primers used were synthesized by the Laboratory of DNA Sequencing and Oligonucleotide Synthesis “oligo.pl” (Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland). One microgram of total RNA primed with anchored oligo(dT)19 primers was used for first-strand synthesis with RevertAid M-MuLV Reverse Transcriptase (Fermentas) according to the manufacturer’s instructions. PCR, using degenerated primers 5′-CAAAGGGACATTACAAGAARTGCATGG-3′ (forward) and 5′-GCTARTGACATYCGWGTCCCGTC-3′ (reverse) constructed on the basis of conserved sequences encoding for ACOs in Lycopersicon esculentum, Nicotiana tabacum, Nicotiana attenuata, Nicotiana glutinosa, Solanum tuberosum (Solanales order) and A. thaliana, was performed in the T3 Thermocycler (Biometra). The total reaction volume contained 2 μL of first-strand cDNA, 2 μL of primer solution (10 μM), 5 μL of buffer B (containing 15 mM Mg2+), 0.5 μL of 50 mM Mg2+, 2 μL of 5 mM dNTP mix and 1.25 U of Blue Perpetual Taq DNA PolymeraseHOT-START (EurX). The reaction mixtures were subjected to the following PCR conditions: 95 °C for 5 min, 1 cycle, 95 °C for 1 min, 55 °C for 45 s, 74 °C for 45 s for 35 cycles, followed by 1 cycle of incubation at 74 °C for 5 min. A 360-bp amplified cDNA fragment was isolated from an agarose gel with the GeneMATRIX Agarose Out DNA Purification Kit (EurX), cloned using the TOPO TA Cloning Kit (Invitrogen) and sequenced by “oligo.pl”. A full-length cDNA coding for PnACO1 was isolated using the BD SMART RACE cDNA Amplification Kit protocol from Clontech. mRNA used in 5′- and 3′-RACE-PCR was isolated with the Oligotex mRNA Mini Kit (Qiagen). 5′-RACE primer 5′-CAGCTTTCTCCACCAGAGCCGGTGCCGG-3′ and 3′-RACE primer 5′-GGTGGACGTTCCTCCCATGCGCCACTCC-3′ were used, and were specific for the PnACO1 cDNA fragment. PCR reactions were performed using the Advantage 2 PCR Enzyme System from Clontech. The reaction products were cloned using the TOPO TA Cloning Kit (Invitrogen). One Shot Mach1-T1 cells (Invitrogen) were plated onto Petri dishes containing S-Gal/LB Agar Blend (Sigma) and 50 μg mL−1 ampicillin (ICN). Ten white colonies were selected and cultured o/n in liquid 2× LB medium containing 50 μg ml−1 ampicillin. Plasmid DNA was isolated with the GeneMATRIX PLASMID MINIPREP DNA Purification Kit (EurX) and sequenced. Sequence data from this article have been deposited at GeneBank under accession number EF127817. Data analyses were performed in ClustalW (http://www.ebi.ac.uk/clustalw), BLAST 2.2 (http://www.ncbi.nlm.nih.gov/ BLAST) and DiAlign (http://www.genomatix.de/cgi-bin/dialign).
Total RNA was isolated with the GeneMATRIX Universal RNA Purification Kit (EurX) according to the manufacturer’s instructions. One microgram of total RNA primed with anchored oligo(dT)19 primers was used for first-strand synthesis with the RevertAid M-MuLV Reverse Transcriptase (Fermentas) according to the protocol. Semi-quantitative (SQ)RT-PCR conditions were optimized to a constitutively expressed actin gene (InACT4), which was used as an internal control. (SQ)RT-PCR analyses were performed with the gene-specific primers: PnACO1 (550 bp) 5′-GAAAAGCTTAATGGCGCTGAGAGAGG-3′ (forward) and 5′-TCGTCTTGGAAGAGGAGGATTATGGCC-3′ (reverse); InACT4 (240 bp) 5′-GAATTCGATATCCGAAAAGACTTGTATGG-3′ (forward) and 5′-GAATTCCATACTCTGCCTTGGCAATC-3′ (reverse). PCR mixtures were made as follows: 2 μL of first-strand cDNA, 2 μL of PnACO1 primer solution (10 μM), 1.5 μL of InACT4 primer solution (10 μM), 5 μL of buffer B (containing 15 mM Mg2+), 0.5 μL MgCl2 (50 mM), 2 μL of 5 mM dNTP mix and 1.25 U of Blue Perpetual TaqHOT-START DNA Polymerase (EurX). T3 Thermocycler parameters for the reaction were set up as follows: 95 °C for 5 min, 1 cycle, 95 °C for 1 min, 58 °C for 45 s, 74 °C for 1 min for 30 cycles, followed by 1 cycle of incubation at 74 °C for 5 min. The computer application used for the analysis was Quantity One (BioRad), and for the calculations and graphs we used MS Office Excel (Microsoft) and SigmaPlot 2001 v. 7.0 (SPSS Inc.).
Isolation of cDNA for the ACC oxidase
A comparison of ACO sequences from A. thaliana (AtACO1 and AtACO2—the only fully described ones) and other plant species in the DiAlign with PnACO1 shows that PnACO1 has 69 % amino acid sequence identity to A. thaliana ACO2 (GB acc. no. NP_176428.1). Based on amino acid sequences, phylogenetic analysis revealed that PnACO1 was very closely related to ACO1 (83 %) from N. attenuata (GB acc. no. AAR99394.1), N. glutinosa (GB acc. no. AAA99792.1) and S. tuberosum (GB acc. no. AAK68075.1) (Fig. b).
PnACO1 expression in vegetative organs of Pharbitis nil seedlings
The highest (four- or fivefold) increase in the PnACO1 transcript level in IAA-treated plants was observed in shoot apices, petioles and hypocotyls, while the lowest (two- or threefold) in cotyledons. Apart from that, in all of the organs studied the level of PnACO1 expression kept dropping following hour 8, to reach a level identified in plants not treated with the hormone at hour 16 (Figs. 2, 3).
PnACO1 expression under LD and SD conditions regulated by light and IAA
In this study, using degenerated PCR primers, and then 5′- and 3′-RCE-PCR techniques, we isolated for the first time ACO cDNA from cotyledons of P. nil. (PnACO1) and determined its sequence. The PnACO1 gene encodes for a protein whose expected molecular mass (34.82 kDa) is similar to that of ACC oxidases identified in other plant species (Hudgins et al. 2006; Hunter et al. 1999; Nie et al. 2001; Vriezen et al. 1999; Zanetti et al. 2002). The largest similarity with respect to the PnACO1 amino acid sequence (83 %) is shown in ACO1 proteins from N. attenuata, N. glutinosa, S. tuberosum, as well as A. thaliana ACO2—69 % (Fig. 1b). Because PnACO1 contains motifs that are evolutionarily conserved and characteristic of ACC oxidases and these motifs are located within the Lys and Arg residues essential for protein activity (Yoo et al. 2006) (Fig. 1a), it can be suspected that this gene encodes for a functional enzymatic protein.
Due to the fact that the ethylene production level in various organs and tissues at different stages of the plant’s development is regulated differently (Tsuchisaka and Theologis 2004b; Yamagami et al. 2003), we studied the expression pattern of PnACO1 in the roots, hypocotyls, petioles, cotyledons and shoot apices of P. nil seedlings cultivated under continuous light, 16-h inductive darkness and after auxin treatment.
The results obtained show that PnACO1 expression occurs in all the vegetative organs studied of P. nil seedlings cultivated under various lighting conditions and after the application of exogenous IAA. The highest level of this gene’s transcript in plants not treated with auxin was observed in cotyledons, whereas the lowest accumulation of its mRNA was found in shoot apices (Figs. 2, 3).
Similar results were obtained from studies on the transcriptional activity of ACC oxidase genes in sunflower seedlings, with the highest ACCO2 expression seen in cotyledons and the lowest in roots (Liu et al. 1997). ACCO1 expression in these organs demonstrated the opposite pattern. At the same time, the level of ACCO3 mRNA observed in cotyledons, roots and hypocotyls was equal, although low. In turn, in potato the highest ACO1 mRNA concentration was found in leaves (Nie et al. 2001). An increased accumulation of NG-ACO1 and NG-ACO3 transcripts was observed in aging leaves of N. glutinosa, while the level of NG-ACO2 mRNA was stable in these organs (Kim et al. 1998). A high level of NG-ACO1 and NG-ACO3 expression was also found in roots and that of NG-ACO2 in stems.
An increased expression of the ACC oxidase gene in P. nil cotyledons (Figs. 2, 3), correlated with a substantial transcriptional activity of PnACS1 and PnACS2 (Frankowski et al. 2009; Kęsy et al. 2010), could be connected with the cotyledons reaching full maturity, or could be the first symptom of their aging. A similar effect was proved in the case of the cotyledons and leaves of Morus alba, in which an increased accumulation of the MaACO1 transcript preceded the occurrence of aging symptoms. Additionally, many papers showed that ethylene can, on its own and as a result of positive feedback, regulate its biosynthesis by increasing the level of expression of genes encoding for ACC oxidases. The existence of this mechanism was proved in the leaves of tomato and melon (Blume and Grierson 1997; Bouquin et al. 1997), etiolated pea seedlings (Peck and Kende 1995; Peck and Kende 1998) or sprouting pea seeds (Petruzzelli et al. 2000).
Application of auxin to P. nil seedlings cultivated both under light and dark conditions causes an increase in the transcriptional activity of the PnACO1 gene in all of the organs studied (Figs. 2, 3). The highest concentration of this gene’s mRNA is observed at hours 4 or 8 (depending on the organ), after which the accumulation of the transcript reaches a level observed in plants not treated with IAA (hour 16). It must also be noted that the maximum level of PnACO1 expression is shifted in time by 2–6 h in relation to the occurrence of PnACS1 and PnACS2 transcripts (Frankowski et al. 2009; Kęsy et al. 2010).
An increased expression of ACC-encoding genes after application of auxins was also observed in other plant species. In potato tubers, auxin has a significant stimulating effect on the accumulation of ST-ACO3 mRNA (Zanetti et al. 2002). In turn, in etiolated hypocotyls of Vigna radiata, IAA increases the expression of VR-ACO2, while having no effect on the level of accumulation of the VR-ACO1 transcript (Yu et al. 1998). Nevertheless, application of another concentration of IAA in further studies resulted in an increased transcriptional activity of both the genes studied (Song et al. 2005).
Our results show that, similarly to ACSs, the expression of genes encoding for ACC oxidases is controlled at various stages of plant development by internal (auxin) and environmental (light) factors and differs in time and space. A high level of PnACO1 expression in P. nil cotyledons treated with auxin shows what role ethylene and its interactions with auxin play in regulating the plant’s growth and development also at early stages of its vegetation. Ethylene produced by shoot apices in dicotyledons limits cell enlargement in young leaves (Kieber et al. 1993; Lee and Reid 1996; Osborne 1991), and—by way of analogy—it could play the same role in other tissues, as well.
Auxin induces the expression of some of the ACC synthase genes (Arteca and Arteca 1999; Frankowski et al. 2009; Tian et al. 2002; Tsuchisaka and Theologis 2004b); therefore, its inhibitory effect on flowering is most probably the result of increased ethylene production (Frankowski et al. 2009; Kęsy et al. 2008). Additionally, the level of accumulation of some ACC synthase transcripts is also regulated by light (Frankowski et al. 2009) and correlates with increased ethylene production (Kęsy et al. 2008). We therefore studied whether the inhibitory effect of auxins on P. nil flowering is correlated with an increase in the transcriptional activity of the ACC oxidase gene, and also determined the level of this gene’s expression in the cotyledons of seedlings cultivated under different photoperiodic conditions.
Based on the results obtained, it can be concluded that both light and auxin control the level of PnACO1 transcript accumulation (Fig. 4). Significant changes in the level of this gene’s mRNA occurred in plants cultivated both under the long- and the short-day conditions. While an increase in the transcriptional activity of PnACO1 at hour 10 in the cotyledons of seedlings cultivated under the LD condition results from the light having been turned on at hour 8, the increase in the gene’s expression observed at the same hour of the diurnal cycle in the cotyledons of seedlings cultivated under the SD conditions seems to be the consequence of an endogenous rhythm. The level of the gene’s expression in plants cultivated under the SD conditions is also increased after the light is turned on (hour 17). Oscillations in the level of PnACO1 mRNA are also observed in the case of IAA-treated plants, whereas an increase in its expression is additionally seen after the light is turned on (hour 17). Nevertheless, the increased level of the gene’s transcriptional activity in IAA-treated plants that is maintained at hours 12–16 seems to be the result of positive feedback, as it correlates with an increased accumulation of PnACS1 and PnACS2 mRNAs (Frankowski et al. 2009; Kęsy et al. 2010). The data presented show clearly that PnACO1 expression is regulated by light and displays diurnal oscillations. Additionally, the increase in PnACO1 concentration after the light is turned on both under the SD and LD conditions is correlated with an increase in PnACS1 transcript accumulation (Frankowski et al. 2009) and an increase in ethylene production (Kęsy et al. 2008).
ACC oxidase genes, whose expression is regulated by light, were also identified in the seedlings of Phaseolus vulgaris (Pidgeon et al. 1997), Stellaria longipes (Kathiresan et al. 1996), Sorghum bicolor (Finlayson et al. 1999) and Vigna radiata (Song et al. 2003). In S. longipes cultivated under the LD conditions, cyclic changes were found in the level of mRNA of the gene encoding for ACC oxidase, which were correlated with the enzymatic activity of the protein (Kathiresan et al. 1996). Cyclic accumulation of SbACO2, as well as cyclic fluctuations in the level of ethylene, was also observed in S. bicolor cultivated under conditions imitating shading (Finlayson et al. 1999). However, changes in the gene’s transcriptional activity did not correlate with the enzymatic activity of its product. In turn, a slight drop in the level of the VR-ACO2 transcript and the constant transcriptional activity of VR-ACO1 were observed in the hypocotyls of etiolated V. radiata seedlings after they were exposed to light (Song et al. 2003).
Application of auxin to the cotyledons of P. nil seedlings at the beginning of the inductive night causes a clear increase in the accumulation of PnACO1 mRNA from the first hours in the dark period (Fig. 4); that increase follows a rise in the accumulation of ACC synthase gene (PnACS1 and PnACS2) transcripts (Frankowski et al. 2009; Kęsy et al. 2010). These data correlate with an increase in ethylene production after application of 1 mM IAA to the cotyledons of P. nil seedlings at the beginning of the inductive night (Kęsy et al. 2008; Wijayanti et al. 1997).
The data presented here and in our previous papers (Frankowski et al. 2009; Kęsy et al. 2008, 2010) clearly show that the inhibitory effect of auxins on P. nil flowering is the result of their stimulating effect on the expression of genes encoding for enzymes involved in ethylene biosynthesis (ACSs and ACOs), which leads to an increase in the concentration of ethylene that inhibits flowering directly.
Although auxin plays a superior role in regulating many plant growth and development processes, still its participation in the photoperiodic induction of flowering occurs through cooperation with other phytohormones, including ethylene. Nevertheless, it cannot be excluded that ethylene has an influence on some other significant factor regulating flowering in this species, e.g., abscisic acid (Wilmowicz et al. 2008). It was shown that auxin-induced increase in ethylene production leads, in turn, to a drop in the accumulation of abscisic acid, whose increased level in the second half of the inductive night is a factor that inhibits P. nil’s transition from the vegetative development stage to generative differentiation.
Emilia Wilmowicz and Kamil Frankowski designed and carried out the experiments, analyzed the data and wrote the manuscript. Agata Kućko carried out the experiments. Paulina Glazińska and Waldemar Wojciechowski were responsible for the preparation of figures and charts. Jan Kopcewicz and Jacek Kęsy helped in preparing the manuscript.
This work was supported by the Nicolaus Copernicus University Grant Program no. 305-B.
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