Carbohydrate mobilization and gene regulatory profile in the pseudobulb of Oncidium orchid during the flowering process
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- Wang, C., Chiou, C., Wang, H. et al. Planta (2008) 227: 1063. doi:10.1007/s00425-007-0681-1
The pseudobulb of Oncidium orchid is a storage organ for supplying water, minerals and carbohydrates to the developing inflorescence. Different patterns of mannan, starch and pectin metabolism were observed in the pseudobulb of three developmental stages by histochemical staining and high performance anion exchange chromatographic (HPAEC) analysis. Copious pectin was strongly stained by ruthenium red in young pseudobulbs demonstrating that mannan and pectin were preferentially accumulated in the young pseudobulb sink at inflorescence pre-initiation stage. Concomitant with the emergence of the inflorescence, mannan and pectin decreased gradually and converted to starch. The starch, synthesized at the inflorescence developing stage, was eventually degraded at the floral development stage. A systematic survey on the subtractive EST (expression sequence tag) library of pseudobulb in the inflorescence pre-initiation stage revealed the presence of five groups of gene homologues related to sucrose, mannan, starch, pectin and other carbohydrate metabolism. The transcriptional level of 13 relevant genes related to carbohydrate metabolism was characterized from pseudobulbs of three different developmental stages. The specific activities of the enzymes encoded by these genes were also assayed. The expression profiles of these genes show that the transcriptional levels largely correlated with the enzyme activities, which were associated with the respective carbohydrate pools. These results demonstrated a novel functional profile of polysaccharide mobilization pathway as well as their relevant gene expression in the pseudobulb of Oncidium orchid during the flowering process.
KeywordsCarbohydrate metabolismEST (expressed sequence tags)InflorescenceOncidiumPseudobulb
High performance anion exchange chromatography
Expression sequence tags
Carbohydrates are important nutritients and energy sources in living organisms. The structural components of the cells are largely made up of carbohydrates. During plant growth and development, photoassimilates produced by the source leaf are translocated toward different sinks for utilization or accumulation. The source leaves accumulate large foliar carbohydrate pools to buffer variations in the rate of photosynthesis and to liberate sufficient sucrose at night to ensure a more or less continuous supply of sucrose for sink organs in many plant species (Geiger 1987). Recent evidence has shown that carbohydrates are able to modulate gene expression. High levels of carbohydrate could inhibit the expression of genes involved in photosynthesis (Van Oosten and Besford 1994), and increase the expression of genes involved in nitrate assimilation, growth, storage and starch remobilization (Thomas and Rodriguez 1994; Baier et al. 2004). It implicates that carbohydrates can perform more complex functions involved in plant growth and development, in addition to structural and nutritional functions (Rook et al. 2006).
During the past 10 years, Oncidium hybrid, such as Goldiana was reported as a C3 shade plant (Hew and Yong 1994). The carbohydrate pool in the pseudobulb during inflorescence development has also been observed (Ng and Hew 1996). Reserved carbohydrates, such as sucrose, glucose and fructose accumulate in the pseudobulb of Gower Ramsey before the initiation of inflorescence (Wang et al. 2003). The sucrose level was transiently elevated to 20 mg g−1 DW at inflorescence developing stage (B1 stage), relative to ∼10 mg g−1 DW at the inflorescence pre-initiation stage (Wang et al. 2003). Then, it declined to ∼10 mg g−1 DW, accompanying the gradual decrease of glucose and fructose level during the late inflorescence developing stage. Recently, a polysaccharide from the pseudobulb of the inflorenscence developing stage was identified and characterized as pure mannan, consisting of more than 95% mannose and a high degree of uniformity in structure (Wang et al. 2006). Although a direct link between the biochemical changes of carbohydrate metabolism in the pseudobulb and in the flowering process has been proposed (Hew and Ng 1996; Wang et al. 2003), the molecular evidence for the metabolic control of the flowering process is still scanty. To understand the accumulation, interconversion and degradation of different polysaccharides in the developmental pseudobulb more precisely, each polysaccharide form should be analyzed individually. As the first molecular approach to demonstrate carbohydrate metabolism and gene regulation correlated with the flowering process in the pseudobulb, we focus on the identification and expression pattern of the functional genes related to carbohydrate metabolism in the pseudobulb of Oncidium hybrid Gower Ramsey. The carbohydrate metabolism in the pseudobulb was monitored by determining the expression level and the enzymatic activity of the relevant genes. In this work, a novel profile of polysaccharide mobilization in Oncidium pseudobulb, which starts from sucrose to mannan, then to starch, was clarified. Furthermore, the functional role of carbohydrate mobilization during the flowering process of Oncidium pseudobulb has been discussed.
Materials and methods
Oncidium Gower Ramsey plants were obtained from Shih-Dong orchid nursery in Taiwan, and were grown in 30 cm diameter pots under controlled conditions at 25–32°C, and a 14 h photoperiod in a glasshouse. Natural irradiation was supplemented with artificial illumination (high pressure sodium lamps of 400 W) to maintain a regular photon flux density of 1,000 μmol m−2s−1.
Histochemical staining of polysaccharides of the pseudobulb
Free-hand sections of the pseudobulb were taken at different stages, as indicated in the legend to Fig. 1. Pectin was localized in the tissues following the method described by Willats et al. (2001). The sections were stained with 0.02% (w/v) aqueous (deionized water) solution of ruthenium red and incubated in the dye for 10 min before microscopic observation. Starch was localized following potassium iodide and iodine staining (Caissard et al. 2004). The sections were directly stained for 15–45 min in 0.5% iodine water containing 1% potassium iodide. The stained sections were observed under a compound microscope (Olympus, IMT-2, Japan) and then the images were photographed.
Extraction and determination of polysaccharides from the pseudobulb
Extraction and determination of starch was performed as described by Wang et al. (2003) using potato starch as standard and 2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS) for the color reaction. Starch was estimated as the amount of glucose released by measuring the absorbance of the reaction at 405 nm.
Mannans were isolated from the pseudobulb according to the method described by Mulimani and Prashanth (2002) with little modification. The pseudobulb powder was stirred in water at 80°C for 5 h and filtered through two layers of Miracloth. The residue was extracted once again with hot water (5 ml/g tissue) for 1 h and filtered. The filtrates were combined and TCA was added to the final concentration of 5% (w/v) while stirring the solution at low speed. The contents were kept at 4°C for 1 h for the precipitation of the protein and centrifuged at 20,000g for 30 min. The supernatant was collected and filtered through two layers of Miracloth. The mannans were precipitated by the addition of chilled ethanol to a final concentration of 80% (v/v) and keeping it at 4°C overnight. The precipitate was collected by centrifugation at 20,000g for 30 min at 4°C. Mannans pellet was washed with a mixture of ethanol and ether (1:1, v/v) and dried in vacuum. The quantity of mannans was determined gravimetrically.
Pectin was isolated from the alcohol insoluble residue of the pseudobulb (Sobry et al. 2005). The fine powder of pseudobulb was dispensed in 80% ethanol (5 ml/g tissue) and boiled for 40 min and filtered (Stancato et al. 2001). The residue was washed twice with 80% ethanol followed by acetone and air dried to get alcohol insoluble residues (AIR). Starch was removed from the AIRs by suspending it in 90% (v/v) dimethylsulfoxide for 16 h at 20°C and centrifuging at 20,000g for 20 min (Stolle-Smits et al. 1999). The pectic polysaccharide was extracted from the starch-free AIR following the method described by Western et al. (2001) with minor modification. The AIR was stirred in 0.5% ammonium oxalate solution (25 ml/g AIR) at 80°C for 1 h and centrifuged at 20,000g for 20 min. The supernatant was collected and ethanol was added five times the volume of the extract to precipitate pectic polysaccharides. The fibrous precipitate was collected by filtration through four layers of Miracloth and vacuum dried and weighed.
Analysis of mannans and pectins by high-performance anion exchange chromatography
Mannans and pectins were dissolved in water and analyzed on high performance anion exchange chromatography (HPAEC) chromatographic equipment (Dionex Corp., Sunnivale, CA, USA) fitted with CarboPac PA-100 anion exchange column (2 × 250 mm; Dionex; with the PA-100 guard column) and electrochemical detector ED-50 (Dionex). Samples were eluted with 0.1 M NaOH (A) and 0.5 M NaOAc in 0.1 M NaOH (B) gradient with the following scheme: 0 min, 90% A and 10% B; 30 min, 10% A and 90% B; 30–40 min, 10% A and 90% B; and 40–50 min, 100%B with a flow rate of 0.25 ml/min. The column was equilibrated to initial conditions for 10 min between each run. Mannan from Saccharomycescervevisiae (Sigma, M7504) and pectin from citrus fruit (Sigma, P9135) were used as standard in HPAEC identification.
Acid hydrolysis and HPAEC-PAD analysis
The monosaccharide composition of pectin and mannans was determined by acid hydrolysis followed by HPAEC-PAD on Carbopak PA10 column (Dionex). At 30°C for 30 min, 10 mg of pectin/mannan was pre-incubated in 0.1 ml of 72% H2SO4 and then the mixture was diluted to 2 N H2SO4 by the addition of 1.1 ml of MilliQ water. The hydrolysis was carried out at 120°C in an autoclave for 1 h. After acid hydrolysis, the mixture was neutralized with 2 N NaOH (prepared from 50% NaOH; Fisher Scientific, Pittsburgh, PA, USA) before HPAEC analysis.
The monosaccharides were analyzed by HPAEC-PAD with ED 50 detector (Dionex) and CarboPak PA-10 analytical column (2 × 250 mm) with PA-10 guard column (Dionex). Acid hydrolysates of mannans were analyzed by eluting the sugars isocratically with 20 mM NaOH for 20 min followed by 200 mM NaOH for 10 min with a flow rate of 0.25 ml/min. The column was equilibrated with 20 mM NaOH for 10 min between each run of samples in a sequence. Acid hydrolysates of pectins were analyzed mainly according to the method described by Talaga et al. (2002) with a convenient modification. The gradients of NaOH and NaOAc were used simultaneously to elute the sugars by mixing NaOH, NaOAc and water appropriately to generate the following gradient of NaOH: 0–15 min, 18 mM; 15–18 min, 18–100 mM; 18–45 min, 100 mM; 45.1–50 min, 18 mM. The simultaneous gradient of NaOAc was: 18–35 min, 0–250 mM; 35.1–45 min, 500 mM. The column was equilibrated for 10 min with 18 mM NaOH between each run of samples in a sequence. The PAD was set to the waveform as specified in the manufacturer’s (Dionex) instructions. Peaks were identified based on the relative retention time of peaks of authentic monosaccharide standards (Sigma). Sugars were quantified after calibration of PAD response to concentration of different sugars.
RNA preparation and Northern-blot assay
The total RNA extraction and Northern-blot analysis were carried out as described earlier (Tan et al. 2005). In brief, the total RNAs of the pseudobulb and its upper leaf were extracted following the pine tree method (Chang et al. 1993). A weight of 10 μg each of total RNAs from the pseudobulb and its upper leaf were separated on 1% agarose/formaldehyde gel and blotted onto nylon membranes (Amersham, Bucks, UK). The EST cDNA fragments were randomly labeled with α-p-32P-dCTP (RediprimeTM II Kit, Amersham) as probe to hybridize the RNA-blotted membranes and the membranes were washed following the standard protocol. The membrane was exposed to fluorescent plate for 12 h (Typhoon 9400, Amersham). Band intensities were normalized with respect to the amount of mRNA loaded.
Extraction and quantification of key carbohydrate metabolizing enzymes from different developmental stages of Oncidium pseudobulb
Plant protein from Oncidium pseudobulb was extracted using P-PER plant protein extraction kit (Pierce Chemical Co., Rockford, IL, USA), according to the manufacturer’s instructions. In brief, 0.5 g of pseudobulb tissue at different development stages was ground into fine powder in liquid nitrogen and dissolved in 2–3 ml of Hepes extraction buffer (pH 7.5). After centrifugation, the supernatant was eluted through Amicon Ultra filter column (Millipore, Bellerica, MA, USA). The concentration of the total protein was estimated by Bradford method (Bradford 1976) and the protein was adjusted to 1 μg per μl. The protein was immediately used for different enzymatic assay or immediately aliquoted and stored frozen at −80°C until further use. The quality of the crude protein was checked by SDS-PAGE resolution and by using RPN8 antibody for Western-blot assay as an internal standard.
Assay of invertase activity
The invertase activity was assayed following the method described by Dreier et al (1998), with some modification. Pseudobulb tissues were ground in liquid nitrogen and suspended in 1 ml of pre-cooled (4°C) separation buffer. The samples were centrifuged at 15,000g for 15–30 min at 4°C and both soluble invertase (supernatant) and wall-bound invertase (pellet) enzymes were assayed separately. The pellets were resuspended in 1 ml of the same solution and used for subsequent enzymatic tests. For each sample, 100 μl of the solution was diluted with 0.2 M Na acetate buffer (pH 4.0 for soluble invertase assays and pH 5.5 for wall-bound invertase assays) to a final volume of 600 μl. The enzymatic reaction was triggered by adding 800 μl of 0.225 M sucrose and supplemented with 50 μl of 1 g/l glucose to avoid oxygen interference at low reducing sugar concentration. An aliquot of 700 μl was pipetted off to a fresh tube as the A1 sample and was incubated at 30°C for 1 h. To the remaining mixture, designated as A0 sample, was immediately added 1 ml of 1% (w/v) DNSA, 0.5 M KOH and 1 M Na/K tartrate, and heated for 10 min at 100°C to stop reaction. The reaction of sample A1 was stopped as described above after 1 h. The absorbance of the reaction samples was measured at 560 nm. The invertase activity was calculated as the difference in reduced sugar levels between the samples A1 and A0. Invertase activities were represented as μg of glucose formed in 1 ml of extract per hour and mg of total protein.
Assay of sucrose synthase activity
The assay mixture contained 50 μg of enzyme along with the substrate 0.1 M sucrose and 500 μM UDP (480 μl) at pH 6.4. The mixture was incubated at 37°C for 60 min. This reaction was terminated by adding 60 μl 1 M Tris buffer (pH 8.8) and boiling the reaction mixture (1 ml) at 80°C for 10 min. To this coupling enzyme reaction buffer containing 0.2 U/ml UDP-glucose dehydrogenase, 0.03 M NAD+ and water were added. NAD+ reduction was measured at 340 nm using a U-3200 Hitachi spectrophotometer (Hitachi, Tokyo, Japan). The initial absorbance at 340 nm prior to the addition of NAD+ to the reaction mixture was taken as reference blank. The reduction in A340 by the addition of NAD+ was noted every minute until no further change in the absorbance was observed.
Assay of GDP-mannose pyrophosphorylase
GDP-mannose pyrophosphorylase (EC 188.8.131.52) was assayed according to Marolda and Valvano (1993). The reaction was started by the addition of Na PPi (freshly prepared) to give a final concentration of 1 mM. The enzyme activity was monitored by measuring the A340 using Hitachi U-3200 spectrophotometer (Hitachi). One unit of enzyme activity was defined as the activity that reduced 1 μM of NADP min−1 at 25°C.
Assay of β-mannosidase
β-Mannosidase enzyme assay was performed by taking 60 μl (50 μg protein) of the extract and incubating it with 90 μl of 2 mM p-nitrophenyl β-d-mannopyranoside (Sigma) in McIlvaine buffer (0.1 M citric acid, 0.2 M Na2HPO4, pH 5.0) for 2 h at 37°C. The reaction was terminated by the addition of 75 μl of 0.2 M aqueous sodium carbonate. The absorbance of the color developed by the reaction is read at 405 nm, considering the extinction coefficient of p-nitrophenyl to be 18,400 to calculate the enzyme activity in terms of pmol min−1 g−1 fresh weight.
Assay of β-1,4-mannose endohydrolase
A known quantity of the enzyme (50 μg) was added to 800 μl of the reaction mixture containing 200 mM acetate buffer (pH 5.0) with 100 mM NaCl, 1% insoluble substrate (AZCL-galactomannan; Megazyme, Bray, County Wicklow, Ireland). The reaction was stirred continually at 37°C. Aliquots (200 μl) were taken at different times and heated for 5 min at 100°C. This was further centrifuged at 12,000g for 5 min and absorbance of the supernatant was read at 595 nm on a microplate reader. Enzyme activity is expressed as Δ595 mg−1 min−1 (Marraccini et al. 2001).
Assay of granule-bound starch synthase (GBSS)
The reaction mixture (200 μl) consisted of 20 μl (50 μg) of the enzyme extract, 100 mM Bicine (pH 8.5), 25 mM potassium acetate, 10 mM DTT, 5 mM EDTA, 1 mg amylopectin and 1 mM ADP [U-14C ] glucose (Amersham) at 1.15 GBq/mol. The reaction mixture was incubated for 10 min at 25°C and the reaction was terminated by heating to 90°C for 2 min. Control assays were terminated immediately after the addition of amylopectin. To the reaction mixture, 3 ml of 75% aqueous methanol containing 1% (w/v) KC1 was added and incubated at room temperature for 5 min. It was further centrifuged at 2,000g for 5 min and the supernatant was discarded. The pellet obtained was redissolved in 0.3 ml water. This washing and resuspension after centrifugation was repeated twice and the radioactivity was determined by liquid scintillation counting (Denyer et al. 1997).
Assay of starch phosphorylase
The reaction mixture contained 50 mM sodium acetate, pH 5.4, 0.3% (w/v) potato soluble starch (Sigma S-2630), 8 mM potassium Glc-1-P, and the enzyme sample in a final volume of 100 μl. After incubation at 37°C for 10 min in a microtiter plate, 200 μl of ferrous sulfate molybdate solution was added to each well, and the inorganic phosphate released was determined according to the procedure of Fiske and Subbarow (1925). The mixture was incubated for 10 min at room temperature and the absorbance at 650 nm was measured by an ELISA reader.
Assay of β-amylase
β-Amylase activity was determined as described by Wang et al. (1995) by adding 50 μg of the protein extract to 50 μl β-amyl reagent (Megazyme) followed by incubation at different time intervals. The reaction was terminated by the addition of 300 μl of 1% Tris to 20 μl of the reaction mixture. After thorough mixing, 150 μl of the solution was pipetted on to a microplate and the A410 was determined. One unit of activity was defined as the quantity of enzyme that released 1 mM of p-nitrophenol min−1 (Gana et al. 1998).
Changes in polysaccharide composition in Oncidium pseudobulb during inflorescence development
Transient accumulation of starch in the pseudobulb of the inflorescence developing stage (starting from B1 to B2 stage) was found by iodine staining (Fig. 1e) and quantification of starch in the pseudobulb (Fig. 2b). This accumulation occurred after the sucrose peak (Wang et al. 2003). Lower level of starch was subsequently observed in the late inflorescence development stage (C stage; Fig. 1f). These data showed that mannan and pectin highly accumulated in the inflorescence pre-initiation stage (A stage), but gradually degraded in the inflorescence emergence and developing stages (B and C stage). On the other hand, starch was notably accumulated in the inflorescence developing stage (B stage), but decreased strongly in the late inflorescence stage (C stage).
Compositional changes in polysaccharides at three different developmental stages of pseudobulb were detected and quantified by HPAEC
Expression pattern of genes related to carbohydrate metabolism in the pseudobulb during three different developmental stages of inflorescence
EST annotations of genes related to carbohydrate metabolism in the Oncidium pseudobulb during flowering stage
Putative identification of gene
Beta-fructofuranosidase 1 (invertase)
GDP-mannose pyrophosphorylase 1
GDP-mannose pyrophosphorylase 2
Beta-1,4 mannan endohydrolase
Granule-bound starch synthase
Alpha 1,4-glucan phosphorylase l isozyme
Pectin esterase 1
Cinnamyl alcohol dehydrogenase
Triosephosphate isomerase, cytosolic (TIM)
Both, sucrose synthase and invertase genes, were actively expressed in young and developing pseudobulb (A stage, Fig. 5a, b). The genes encoding mannan biosynthetic enzymes such as mannose-6-phosphate isomerase, GDP-mannose pyrophosphorylase-1, GDP-mannose pyrophosphorylase-2 and mannosyltransferase were active in the pseudobulb of inflorescence pre-initiation (A stage, Fig. 5c–f), and early inflorescence developing stage (B1 stage). Genes for mannan degrading enzymes such as β-mannosidase and β-(1, 4)-mannan endohydrolase, however, were active in the middle inflorescence developing stage of the pseudobulb (B2 stage, Fig. 5g, h). The expression level of granule-bound starch synthase peaked at the inflorescence-developing stage (B1 and B2 stage, Fig. 5i). However, the mRNA transcripts of starch phosphorylase were abundant in the later inflorescence stage of the pseudobulb (B2 and C stage; Fig. 5j). On the other hand, the expression of pectin esterase, polygalacturonase and pectate lyase genes were simultaneously observed in the pseudobulb inflorescence pre-initiation and in the early stage of inflorescence development (Fig. 5k–m).
Enzyme activity of the key carbohydrate metabolizing enzymes in the pseudobulb during three different developmental stages of inflorescence
The pseudobulb of Oncidium orchid is a strong sink for partitioning the photosynthates during vegetative development. During its growth, the photosynthates produced in the upper younger leaves are transported to the developing pseudobulb sink. In our present study, carbohydrates in Oncidium pseudobulb of different developmental stages were analyzed by histochemical staining and HPAEC. Polysaccharides of mannan and pectin are extremely abundant in young and developing pseudobulbs (Figs. 1, 2, 3) at the inflorescence pre-initiation stage (designated as A stage). It is well known that plants at the vegetative stage always convert sugar photosynthates into polysaccharides and accumulate them in sinks as nutritional and energy sources. As the inflorescence initiates from the pseudobulb base (designated as B stage), Oncidium switches its life cycle from the vegetative stage to the reproductive stage. Concurrently with inflorescence initiation, the polysaccharides of mannan and pectin start to mobilize in the pseudobulb. This results in the appearance of starch grains from the early inflorescence developing stage (B1 stage). Concomitant with the growth and development of inflorescence (B2 stage), starch grains are gradually degraded for energy supply and eventually used up in the late inflorescence stage (designated as C stage) during which the floral organs are complete developed.
In accordance with the previous reports describing that sucrose, mannan and starch occurred in the carbohydrate pool of the pseudobulb in the inflorescence pre-initiation stage (Wang et al. 2003, 2006), our present results now demonstrate carbohydrates starting from sugar to mannan/pectin and to starch in the pseudobulb during inflorescence development. At stage A of the inflorescence pre-initiation, high-level activities of invertase and sucrose synthase function to cleavage sucrose for mannan and pectin synthesis (Fig. 6a, b). A drop-off of sucrose synthase and invertase activities subsequently occurs in B1 stage of the pseudobulb (Fig. 6a, b), concomitant with a transient peak of the sucrose level (Wang et al. 2003). Thus, the transient accumulation as well as degradation of sucrose at an early inflorescence stage correlates with the transient decline in sucrose synthase activity, while invertase activity remains low. The starch grains start to accumulate at the B1 stage and peak at B2 stage (Fig. 2b). This metabolic network implicates that sucrose plays a functional role to regulate starch biosynthesis during the inflorescence developmental stage of pseudobulb. It is well known that elevated sugars can up-regulate genes for enzymes involved in starch biosynthesis, including granule-bound starch synthase and branching enzyme (Salehuzzaman et al. 1994). In our previous work (Wang et al. 2003), sucrose content was monitored at an elevated level during early inflorescence developing stage (correspondent to B1 stage). Coincidentally, the high expression level of starch synthase was detected at the same stage (Figs. 5i, 6f), and starch grains were apparently observed (Figs. 1e, 2b). This confirms that a highly elevated sucrose level precedes the start of starch biosynthesis.
Even though the Oncidium orchid has become a valuable commercial product in the flower market, comprehensive information concerning its physiology and molecular biology by systematic research is still scarce and is awaited. In this regard, in addition to the above-mentioned biochemical data, our subtractive EST data bank provided an informative knowledge base for functional gene studies (Tan et al. 2005). The subtractive EST genes, which are preferentially expressed in pseudobulb could be functionally categorized into sucrose-, mannan-, starch-, and pectin-metabolizing groups in this work. Further, Northern-blot data demonstrate that the expression pattern of each gene varied, but the expression levels correlated with the pseudobulb and inflorescence development at a relative level (Fig. 5). The gene transcriptional patterns were confirmed/complemented by biochemical activity assay of the gene-encoded enzymes (Fig. 6). These molecular data indicate that carbohydrate mobilization in flowering pseudobulb involves extensive transcriptional regulation of sucrose, mannan, pectin and starch metabolic pathways. This metabolic gene regulatory network and the associated carbohydrate mobilization are a novel finding in this orchid plant.
The presence of an apparent metabolic gene regulatory network for carbohydrate metabolism in the Oncidium pseudobulb during the flowering process
The pectin metabolism is also linked to the mobilization of sucrose in the pseudobulb before initiation of inflorescence (Fig. 7) as shown by the increased pectin content in this stage (Figs. 1, 2c). A qualitative change in pectin was more prominent in the pseudobulb than in mannan during inflorescence development (Figs. 3b, 4). This qualitative change in pectin is probably brought about by the action of pectin esterase (pectin methylesterase) into demethylated pectin. The mRNA transcripts for pectin modifying enzymes were at a higher level during the inflorescence pre-initiation stage (Fig. 5k, l) and early inflorescence developing stage (Fig. 5m). This modification was evident on ruthenium red staining (Fig. 1a–c).
Physiological significance of the sucrose–mannan–starch metabolic pathway in the Oncidium orchid
The metabolic network occurring in the Oncidium orchid appears to be a complicated cycle. An intermediate polysaccharide reservoir of mannan seems a redundant step in the biological utilization. However, mannan has also a function in stress resistances to drought and pathogen infection.
Photosynthetic production of sugars and starch is regulated by the demand for a sink. Carbohydrate mobilization is required for the transition from vegetative to reproductive growth in most plants. Some evidence has been provided that sucrose is an early and essential component of flowering stimulus in most species. Soluble sugars have been implicated in the regulation of developmental processes, such as the timing of flowering (Bernier et al. 1993). The increased supply of sucrose triggers the start of its activation, suggesting that the extra sucrose plays a signaling role in stimulating flowering (Corbesier et al. 1998), and the extra sucrose usually comes from reserve (starch) mobilization. In the Oncidium pseudobulb, starch mobilization was monitored at the inflorescence developing stage (B2 stage) for the energy supply of floral development. Although starch mobilization is thought to be critical for floral induction in most plants (Corbesier et al. 1998), it seems that the transition mechanism from vegetative to reproductive growth in Oncidium is not correspondent to the onset of starch mobilization in the pseudobulb. This suggests that the mechanism of irregular flowering time of Oncidium might be affected/ controlled by some more factors rather than sucrose alone.
In addition to this, we have observed high levels of ascorbic acid in the pseudobulb just before inflorescence initiation (results not shown). Previous reports have shown that l-ascorbate precursor, galactono-1,4-lactone accumulates in the pseudobulb of Oncidium (Wang et al. 2003). In the present context, it should be inferred that the expression of GDP-mannose pyrophosphorylase (Fig. 5d and e) is involved in the production of GDP-mannose, a key intermediate in the production of l-ascorbic acid through the Smirnoff-Wheeler biosynthetic pathway (Smirnoff et al. 2001) in the early stage of inflorescence development. In addition to its antioxidant action, l-ascorbic acid plays a role in the non-enzymatic solubilization of pectins into OGAs (Dumville and Fry 2000, 2003), and causes the compositional change of pectin. This appears coincident with the finding of the substantial alternation of pectin composition (Figs. 3b, 4). OGAs have been considered as non-traditional plant growth regulators with the ability to induce flowering along with other functions (Creelman and Mullet 1997; Etzler 1998). OGAs are also implicated in the induction of oxidative burst as a defense response of plants to pathogen attack, ethylene response and fruit ripening (Dumville and Fry 2000). Although OGAs have not been detected in this work, the physiological function is still predictable.
This numerous interactions and intersections, which occur are potentially important for the modulation and balancing of various inputs from different signaling cascades so that plants can integrate all this information to execute the proper developmental responses. The differences in the transcript levels and enzymatic activity are important, but for many biological processes in plants the interrelated metabolic pathways seem to be enigmatic and provide a better understanding of the underlying mechanisms. Therefore, the regulatory metabolic network occurring in Oncidium pseudobulbs could be a source of developmental signals for inflorescence induction. Further investigation of the specificity and physiological significance of this carbohydrate metabolic gene regulatory network and the carbohydrate mobilization in the pseudobulb tissue of Oncidium orchids is needed.
The authors are grateful to the National Science Council, Taiwan for the financial support granted to Professor Kai-Wun Yeh under the project NSC-93-2317-3-002-012. The authors are also grateful to Professor Zhang, R-H and Professor Wang A-Y, at the Department of Biochemistry, NTU, for their assistance in the enzymatic activity assays of starch phosphorylase and sucrose synthase, respectively. Appreciation is expressed to Associate Professor Hsieh H-L, at the Institute of Plant Biology, NTU for his kind provision of RPN8 antibody.