JXB Advance Access originally published online on July 28, 2003
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Journal of Experimental Botany, Vol. 54, No. 390, pp. 2189-2191,
September 1, 2003
© 2003 Oxford University Press
Cloning of a sucrose-phosphate synthase gene highly expressed in flowers from the tropical epiphytic orchid Oncidium Goldiana
Received 17 December 2002; Accepted 13 June 2003
Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260
* To whom correspondence should be addressed. Fax: +65 6779 2486. E-mail: dbshewcs{at}nus.edu.sg
Abbreviations: SPS, sucrose-phosphate synthase; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase-polymerase chain reaction.
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Sucrose-phosphate synthase (SPS) is one of the key regulatory enzymes in carbon assimilation and partitioning in plants. It plays a crucial role in the production of sucrose in photosynthetic cells. The cloning and expression analysis of a full-length cDNA encoding SPS from tropical epiphytic orchid hybrid Oncidium Goldiana are reported here. The cDNA designated as sps1 is 3820 bp in length with an open reading frame of 3183 bp encoding 1061 amino acids. The deduced amino acid sequence of O. Goldiana sps1 shows 56% and 69% homology with those of maize SPS and spinach SPS, respectively. The high level expression of O. Goldiana sps1 in the flower suggests that it might play an important role in flowering. Growth under higher irradiance and elevated CO2 leads to an accumulation of the sps1 transcript in the photosynthetic leaves. It appears that SPS gene expression in photosynthetic leaves is associated with the leaf photosynthetic rate.
Key words: Gene expression, Oncidium Goldiana, quantitative RT-PCR, sucrose-phosphate synthase.
Sucrose-phosphate synthase (SPS) is a key regulatory enzyme involved in carbon assimilation and partitioning of photoassimilate between sucrose and starch in plants (Huber and Huber, 1996; Koch, 1996). To date, most of the studies on the role of SPS in the regulation of sucrose synthesis are at the biochemical and physiological levels. In recent years, more and more interest has been centred on the regulation of SPS at the molecular level. However, full-length or fragment SPS cDNAs have been isolated from fewer than 20 plant species so far. Gene expression studies have been performed in fewer than 10 plant species. Many of these studies have been confined to the study of the relative gene expression level in different plant organs. In general, SPS mRNA appears to be greater in source leaves than in sink leaves (Klein et al., 1993; Valdez-Alarcón et al., 1996). The accumulation of SPS mRNA during leaf development coincided with the leaf transition from sink to source status (Klein et al., 1993; Cheng et al., 1996). SPS mRNA increases in detached sugar beet leaves (Hesse et al., 1995) and Vicia faba cotyledons (Weber et al., 1996) when cultured in vitro with glucose-containing media. However, the SPS transcript slightly decreases in detached sugar beet leaves when cultured with sucrose (Hesse et al., 1995), while it remained unchanged in sugar beet seedlings grown on sucrose-containing media (Kovtun and Daie, 1995). It has also been reported that SPS mRNA increases following transfer from low irradiance to higher irradiance (Klein et al., 1993; Cheng et al., 1996) and exposure to low temperature (Reimholz et al., 1997; Langenkämper et al., 1998).
So far there has been no report on SPS regulation in any tropical epiphytic plant at the molecular level. In a previous study, it was found that leaf SPS activity increases in orchid leaves grown at elevated CO2 (Li et al., 2001). The authors are now interested in whether there is a corresponding increase in leaf SPS mRNA level in these CO2-enriched plants. To do this, the gene encoding SPS has been isolated.
Adult mericloned plants of O. Goldiana were obtained from a local nursery. The plants were maintained in pots of sand in controlled environmental growth chambers. The growth chamber provided 150–200 µmol m–2 s–1 of photosynthetically active radiation at leaf height (THORLUX lamp, 400 W, 240 V, 50 Hz, made in UK) [O. Goldiana is a shade plant, light saturation for leaf photosynthesis occurs around 90 µmol m–2 s–1 (Hew and Yong, 1994)], 70–80% relative humidity, 28/25 °C day/night temperature, and a 12 h photoperiod. Half-strength Hoagland’s solution B was used as the culture medium. Unless otherwise stated, all samples were taken at 14:00 local time and immediately frozen in liquid nitrogen.
Total RNA was isolated from different tissues at different developmental stages using NucleoSpin RNA plant kit (Macherey-Nagel, Düren, Germany). To obtain cDNA encoding SPS, a 1000 bp fragment was first obtained by RT-PCR with the following primers: SPS1: 5'-GGT CGT GAT TCT GAT ACT GGT GGT CAG GT-3'and SPS2: 5'-TGG ACG ACA TTC TCC AAA TGC TTT GAC-3'. RT-PCR was performed with the one-step RT-PCR kit (BD Biosciences, Palo Alto, CA, USA) according to the manufacturer’s instructions. The conditions for PCR amplification were as follows: 35 cycles at 94 °C for 30 s, 65 °C for 30 s and 68 °C for 1 min. The amplified fragment was cloned into the pTZ57R vector with the InsT/Aclone PCR Cloning Kit (Fermentas, Vilnius, Lithuania). The identity of the amplified product was confirmed by sequencing.
The following gene-specific primers were designed from the sequence of the 1000 bp fragment to perform 5' and 3' RACE using the SMART RACE cDNA kit (BD Biosciences) according to the manufacturer’s instructions. GSP1-SPS: 5'-CCC ATG ATT CTT GCT CTT GCT CGG CCT GA-3'; GSP2-SPS: 5'-TCC AGG GGG AAT CAC AAC CAT ACG AGG CAT-3'. The 5' RACE and 3' RACE products were cloned into the pTZ57R vector and sequenced. Full-length cDNA was generated by Long-Distance PCR with primers designed from the 5' end of the 5' RACE product sequence and 3' end of the 3' RACE product sequence. The template for this final PCR reaction was from the original first-strand RT-PCR step. Isolated cDNA clones were sequenced using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Applied Biosystems, Foster City, California, USA) on the ABI PRISM 377 DNA sequencer (Perkin-Elmer Applied Biosystems).
The isolated SPS cDNA designated as sps1 is 3820 bp in length with an open reading frame of 3183 bp encoding 1061 amino acids. The deduced amino acid sequence of O. Goldiana sps1 shares 75% identity with that of potato sps (X73477 [GenBank] ), 71% with spinach sps (L04803 [GenBank] ), 56% with maize sps (M97550 [GenBank] ), and 54% with rice sps1 (U33175 [GenBank] ) (Table 1). Recent phylogenetic analyses on plant SPS genes have demonstrated that there are three families of SPS genes present in higher plants (Langenkämper et al., 2002). According to this study, O. Goldiana sps1 falls into family A, which consists of the largest number of plant SPS genes, but with only one other monocotyledonous plant SPS gene, sugarcane sps2. Family A genes are more frequently expressed than other family members (Langenkämper et al., 2002). This is consistent with the present study: O. Goldiana sps1 mRNA was expressed in all the organs analysed (Fig. 1A). These results suggested that sps1 might play a housekeeping role in O. Goldiana. However, the expression level of O. Goldiana sps1 mRNA was not closely correlated with the SPS enzyme activity in different organs (Fig. 1). This result might be due to (1) other isoforms of SPS which have distinct patterns of expression might exist in O. Goldiana as suggested by Langenkämper and co-workers (2002); (2) SPS activity was modified by the phosphorylation of SPS protein (Huber and Huber, 1996).
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To study the regulation of SPS gene expression by sugars, micropropagated O. Goldiana plantlets with 4–5 leaves obtained from a local nursery were grown in GA7 boxes with half-strength Hoagland’s solution B solidified with agar and supplemented with 4% sucrose or 4% glucose for 6 d. To examine the effect of light intensity on the expression of the SPS gene, plants with a young shoot were grown under different light conditions of 200 and 20 µmol m–2 s–1 for 12 d. To study the regulation of SPS gene expression by CO2 enrichment, plants with a young shoot were grown at (1) elevated CO2 with a CO2 concentration about double ambient (within the range of 760±75 µl l–1 for 80% of the time) or (2) ambient CO2 (about 380 µl l–1) fed with ambient air. In the elevated CO2 treatment carbon dioxide was added day and night. The CO2 concentration was monitored using an Infrared CO2 Controller (Fuji Electric Co., Ltd., Tokyo, Japan). After 12 d of growth, youngest fully expanded leaves were harvested for RNA isolation.
For the quantitative competitive RT-PCR, a 935 bp fragment of O. Goldiana SPS was obtained by RT-PCR using the following two primers. SPS-QRT1: 5'-GAG GCC GCC GAG ATG CTA CTG CTG ATA TG-3' and SPS-QRT2: 5'-TGA AAC GAC CGT AGC AAC TGA CAC CTC GCT-3'. The fragment was cloned into the pGEM-T-Easy vector containing a T7 promoter (Promega, Madison, Wisconsin, USA). The plasmid containing the 935 bp fragment was digested with Sma I (Promega) and a 456 bp fragment was removed from the plasmid. The linearized plasmid was recovered from the agarose gel and subjected to re-ligation. The resulting plasmid contained a 479 bp fragment of O. Goldiana SPS. RNA was transcribed from the plasmid in vitro using T7 RNA polymerase (Fermentas). Differing amounts of the in vitro transcribed RNA were added to a set amount of sample RNA as an internal competitive standard (Bonino et al., 2001). RT-PCR was then carried out using the two primers SPS-QRT1 and SPS-QRT2 with the One-Step RT-PCR kit (BD Biosciences). Because there were two kinds of RNA template, sample RNA and internal competitive RNA in each RT-PCR reaction, two RT-PCR products with different lengths could be obtained with one pair of primers. One product with 935 bp could be amplified from the sample RNA and another with 479 bp could be amplified from the competitive RNA. When the amount of SPS mRNA in the sample RNA was equal to the amount of competitive RNA, equal amounts of two PCR products could be amplified from them and two bands with the same signal strength could be visualized when the RT-PCR products were separated on agarose gels.
There are two unique features with regard to the expression patterns of O. Goldiana sps1. First, the expression level of O. Goldiana sps1 is extremely low (less than 1 pg per 0.5 µg total RNA) regardless of the plant organ (Fig. 1B). The low level expression of O. Goldiana sps1 in the leaves might be due to the fact that O. Goldiana is a very slow-growing plant with a very low photosynthetic rate (Li et al., 2001). This is supported by the findings that the expression levels of SPS genes were much higher under higher irradiance than that under lower irradiance (Fig. 2B) (Klein et al., 1993; Cheng et al., 1996). The photosynthetic rate of O. Goldiana is 0.5–1.5 µmol CO2 m–2 s–1 when measured under ambient CO2, which is only about 10% of that of other C3 plant species which is 10–20 µmol CO2 m–2 s–1 (Salisbury and Ross, 1992). The expression level of SPS genes in photosynthetic leaves seems to be closely associated with leaf photosynthetic rate.
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Second, the highest level of expression of O. Goldiana sps1 was found in the flowers. The steady-state level of sps1 mRNA in the flowers is more than four times as much as that in the young leaves. This is interesting because no similar expression pattern has been found in other plant SPS genes. Most of the known SPS genes are predominantly expressed in photosynthetic tissues or tuberous roots. There are two possible explanations for this unique expression pattern. First, SPS in the flower of O. Goldiana might contribute to the long life of the flower (Hew and Yong, 1997). SPS might function to provide the respiratory substrate for the flower. Second, SPS in the flower of O. Goldiana might function to provide nectars to attract insects for pollination. It has been found that nectars of Oncidium contain sucrose, glucose and fructose (Arditti, 1992).
The expression of O. Goldiana sps1 was not affected by incubation with 4% sucrose or 4% glucose (Fig. 2A). Similar results have been reported for sugar beet seedlings grown on media containing 90–300 mM sucrose or glucose (Kovtun and Daie, 1995). These results are contrary to in vitro studies with detached leaves or cotyledons (Hesse et al., 1995; Weber et al., 1996). In these studies, feeding glucose to the detached organs significantly increases the accumulation of the SPS transcript. These results suggest that different mechanisms of gene regulation might be involved in whole plant and in vitro studies.
Elevated CO2 significantly increased the steady-state sps1 mRNA level in the leaves of O. Goldiana. This is consistent with the up-regulation of leaf SPS activity in CO2-enriched plants (Li et al., 2001). Elevated CO2 markedly increased the leaf photosynthetic rate of O. Goldiana (Li et al., 2001). These results further support the hypothesis that the levels of SPS gene expression in photosynthetic leaves might be associated with the photosynthetic rates. Further work is needed to verify this point.
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