Responses of carboxylating enzymes, sucrose metabolizing enzymes and plant hormones in a tropical epiphytic CAM orchid to CO2 enrichment

Authors


Correspondence: Choy Sin Hew. Fax: +65 7792486; E-mail: dbshewcs@nus.edu.sg

Abstract

After exposure to a doubled CO2 concentration of 750 µL L−1 for 2 months, average relative growth rate (RGR) of Mokara Yellow increased 25%. The two carboxylating enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPCase), responded differently to CO2 enrichment. There was a significant daytime down-regulation in Rubisco activity in the leaves of CO2-enriched plants. However, PEPCase activity in CO2-enriched plants was much higher in the dark period, although it was slightly lower during the daytime than that at ambient CO2. Leaf sucrose–phosphate synthase (SPS) and sucrose synthase (SS) activities in CO2-enriched plants increased markedly, along with a night-time increase in total titratable acidity and malate accumulation. There was a remarkable increase in the levels of indole-3-acetic acid (IAA), gibberellins A1 and A3 (GA1+3), isopentenyladenosine (iPA) and zeatin riboside (ZR) in the expanding leaves of plants grown at elevated CO2. It is suggested that (1) the down-regulation of Rubisco and up-regulation of SPS and SS are two important acclimation processes that are beneficial because it enhanced both photosynthetic capacity at high CO2 and reduced resource investment in excessive Rubisco capacity; (2) the increased levels of plant hormones in CO2-enriched M. Yellow might play an important role in controlling its growth and development.

Abbreviations
ABA

abscisic acid

AGPase

ADP-glucose pyrophosphorylase

GA1+3

gibberellin A1 and A3

IAA

indole-3-acetic acid

iPA

isopentenyladenosine

PEPCase

phosphoenolpyruvate carboxylase

RGR

relative growth rate

Rubisco

ribulose-1,5-bisphosphate carboxylase/oxygenase; SPS, sucrose-phosphate synthase

SS

sucrose synthase

UGPase

UDP-glucose pyrophosphorylase

ZR

zeatin riboside

Introduction

Atmospheric CO2 concentration has a significant impact on leaf photosynthesis in C3 plants (Lawlor & Mitchell 1991; Stitt 1991; Bowes 1993). Elevated CO2 concentration usually results in a significant increase in leaf photosynthesis and vegetative growth. In a survey of 60 experiments, it was found that growth in elevated atmospheric CO2 increased photosynthesis 58% compared with the rate for plants grown at ambient CO2 (Drake, Gonzalez-Meler & Long 1997). Summarizing data from the literature on 156 species grown under twice-ambient CO2 concentration, Poorter 1993) noted an average 22% increase in dry matter accumulation in C4 species. Photosynthesis by C4 species is more readily saturated as atmospheric CO2 concentration rises (Lawlor & Mitchell 1991), because their main carboxylating enzyme phosphoenolpyruvate carboxylase (PEPCase) utilizes HCO3 as its substrate. PEPCase is insensitive to O2, and little photorespiration is observed in C4 plants. CAM plants use PEPCase for the initial CO2 fixation, so their responses to CO2 enrichment could be similar to those of C4 plants if PEPCase were saturated at the current atmospheric CO2 concentration (Ting 1994; Winter & Smith 1996). However, many CAM plants also take up CO2 during the daytime, especially under well-watered conditions, when the initial CO2 fixation is by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Nobel 1988). These facultative CAM species might respond more to elevated atmospheric CO2 concentration (Ting 1994).

Sucrose-phosphate synthase (SPS) is a key regulatory enzyme involved in the partitioning of photo-assimilate between sucrose and starch in leaves (Stitt & Sonnewald 1995; Winter & Huber 2000). Sucrose synthase (SS) activity has been shown to play a crucial role in plant metabolism (Déjardin et al. 1997; Sturm & Tang 1999). Of the limited studies on responses of CAM plants to CO2 enrichment so far (Drennan & Nobel 2000), few has been done on a tropical epiphytic CAM species, and the effects of CO2 enrichment on growth, metabolite levels, carboxylating enzymes and sucrose-metabolizing enzymes have not been collectively studied in an integrated manner.

It has been established that plant hormones including auxins, gibberellins, cytokinins and abscisic acid (ABA) are involved in controlling developmental events within apical meristems such as cell division, cell elongation and protein synthesis. Unfortunately, the levels of different plant hormones in high-CO2-grown plants remain largely unstudied. Recently, a significant increase in cytokinins content in low-nitrogen leaves under CO2 enrichment was found in cotton plants (Yong et al. 2000). Mokara Yellow is a tropical epiphytic perennial CAM hybrid. The objectives of this study are to examine (1) how CO2 enrichment affects growth and CAM activity in M. Yellow; (2) how Rubisco and PEPCase respond to CO2 enrichment; (3) how SPS and SS respond to CO2 enrichment; and (4) how CO2 enrichment affects plant hormone levels in M. Yellow.

Materials and methods

Plant materials

Plants with 12–15 leaves of Mokara Yellow were obtained from a local nursery. They were planted in pots of sand in controlled environment 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, Redditch, UK), 70–80% relative humidity, 30/25 °C day/night temperature and 12 h photoperiod. Plants were watered daily with half strength Hoagland’s solution B. Unless otherwise stated, the youngest fully expanded leaves were harvested under growth CO2 conditions, frozen in liquid nitrogen, and stored at −80 °C. The frozen samples were ground to a fine powder in a mortar with liquid nitrogen, and aliquots were used for analyses.

Experimental design and CO2 monitoring

Two different treatments were used: (1) high CO2 with a CO2 concentration about double ambient (760 ± 200 µL L−1); (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 Infrared CO2 Controller (Fuji Electric Co., Ltd, Tokyo, Japan).

Growth, titratable acidity and malate measurement

Growth analysis was performed according to Beadle (1993). The mean relative growth rate (RGR) is measured by the following equation: RGR = (ln W2 − ln W1)/(t2 − t1). For the measurement of total titratable acidity, about 0·5 g leaves were cut into strips and extracted twice in 30 mL boiling distilled water. The pooled extract was made up to 200 mL and the acidity determined by titration with 0·01 N NaOH to an end point of pH 7·0. Titratable acidity is expressed as micro-equivalents of acid per gram fresh weight of tissue. Malate content was determined enzymatically as described by Gutmann & Wahlefeld (1974).

Rubisco and PEPCase extraction and assay

Rubisco extraction and activity assay were carried out according to Vu, Allen & Bowes (1983) and optimized for M. Yellow. About 0·5 g (fresh weight) of liquid nitrogen-frozen leaf samples were ground to a fine powder in liquid nitrogen with a mortar and pestle. As soon as the liquid nitrogen had evaporated, 5 mL of extraction medium consisting of 100 mm Bicine-KOH, 5 mm DTT, 20 mm 2-mercaptoethanol, 10% glycerol and 2% polyvinyl polypyrollidone was added and quickly homogenized. The pH of the extraction medium was optimized as 8·8, 8·5, 8·2, 8·2 and 8·5 for samples harvested at 0800, 1200, 1600, 2000 and 2400 h local time, respectively. The homogenate was immediately centrifuged for 1 min at 11 000 g and the supernatant was immediately used for assays. Twenty microlitre aliquots of the supernatant were used for the determination of total soluble protein in the extract. The reaction mixtures contained 50 mm Bicine-KOH (pH 8·0), 5 mm DTT, 15 mm MgCl2, 0·5 mm RuBP, 20 mm NaH14CO3 and 0·1 mL extract in a total volume of 0·5 mL in 5 mL scintillation vials. The reaction mixtures were flushed with N2 for 10 min before adding extract and NaH14CO3. Initial Rubisco activity was determined by adding 0·1 mL fresh extract to a reaction mixture that is otherwise complete and terminating the reaction after 60 s with 0·1 mL of 6 N HCl. Total Rubisco activity was determined by adding the same volume of extract to a reaction mixture containing all constituents except RuBP, leaving the mixture to incubate at 25 °C for 5 min, then adding the RuBP. To remove unfixed 14CO2, vials were flushed with air overnight. The remaining dried pellets were resuspended in 0·2 mL of 50% ethanol and then 5 mL scintillation cocktail (Ecoscint; National Diagnostics, Atlanta, GA, USA) was added. Radioactivity was measured with a Beckman LS 6000 liquid scintillation counter (Beckman Instruments, Fullerton, CA, USA).

PEPCase activity was assayed in the same extract as Rubisco following a similar procedure. The assay mixture contained 100 mm Bicine (pH 8·0), 10 mm MgCl2, 6 units of malic dehydrogenase, 25 µm NADH and 10 mm NaH14CO3. Reaction was initiated by adding 1 mm phosphoenolpyruvate and was terminated after 2 min at room temperature by adding 0·1 mL of 6 N HCl (Israel & Nobel 1994).

SPS and SS extraction and assay

Assays were performed according to Huber & Huber (1991) with slight modification. About 0·5 g (fresh weight) of liquid nitrogen-frozen leaf samples were ground to a fine powder in liquid nitrogen with a mortar and pestle. Immediately after the evaporation of liquid nitrogen, 5 mL extraction buffer containing 100 mm MOPS-NaOH, 5 mm MgCl2, 1 mm EDTA, 2·5 mm DTT, 0·1% (v/v) Triton X-100 and 10% glycerol was added and homogenized. The pH of the extraction medium was optimized as 7·8, 7·6, 7·5, 7·5 and 7·8 for samples harvested at 0800, 1200, 1600, 2000 and 2400 h local time, respectively. Homogenates were centrifuged at 11 000 g for 60 s. Supernatants were desalted immediately by centrifugal filtration on Sephadex G-25 columns equilibrated with 50 mm MOPS-NaOH (pH 7·5), 5 mm MgCl2, 2·5 mm DTT.

In the limiting assay of SPS, reaction mixtures (100 µL) contained 50 mm MOPS-NaOH (pH 7·5), 15 mm MgCl2, 2 mm fructose 6-P, 12 mm glucose 6-P, 10 mm UDPG, 10 mm Pi (an inhibitor) and 40 µL desalted extract. In the saturating (or Vmax) assay of SPS, Pi was omitted and the concentration of fructose 6-P was increased to 10 mm and glucose 6-P to 40 mm. Reactions were terminated with 100 µL 30% KOH after being incubated at 25 °C for 10 min. Tubes were placed in boiling water for 10 min to destroy any un-reacted fructose or fructose 6-P. After cooling, 3 mL of a mixture of 0·14% anthrone in 13·8 m H2SO4 was added and incubated in a 40 °C water bath for 20 min. After cooling, the colour development was measured at 620 nm. The activation state of SPS is calculated as velocity in the limiting assay expressed as percentage of the Vmax activity.

The procedure for the sucrose synthase assay (measured in the sucrose synthesis direction) was identical to that of SPS except that the reaction mixtures contained 10 mm fructose and did not contain fructose 6-P or glucose 6-P.

ADP-glucose pyrophosphorylase (AGPase) and UDP-glucose pyrophosphorylase (UGPase) extraction and assay

AGPase and UGPase extraction and assay were performed according to Smith, Bettey & Bedford (1989). About 0·5 g (fresh weight) of liquid nitrogen-frozen leaf samples were ground to a fine powder in liquid nitrogen with a mortar and pestle. As soon as the liquid nitrogen had evaporated, 5 mL of extraction medium consisting of 100 mm Hepes, 5 mm DTT and 10% glycerol was added and homogenized. The pH of the extraction medium was optimized as 8·5, 8·2, 8·0, 8·0 and 8·2 for samples harvested at 0800, 1200, 1600, 2000 and 2400 h local time, respectively. Homogenates were centrifuged at 10 000 g for 1 min at 4 °C. Supernatants were desalted on Sephadex G-25 columns equilibrated with extraction medium and were kept on ice prior to assay. Twenty microlitre aliquots of the supernatant were used for the determination of total soluble protein in the extract. AGPase assay mixtures contained, in a total volume of 1 mL, 75 mm Hepes (pH 8·0), 1·5 mm sodium pyrophosphate, 5 mm MgCl2, 0·4 mm NAD, 1 mm ADP glucose, 5 units of glucose-6-phosphate dehydrogenase (NAD-linked, from Leuconostoc mesenteroides), 2 units of phosphoglucomutase and 50 µL extract. The reaction was initiated with sodium pyrophosphate and was monitored spectrophotometrically at 340 nm. UGPase assay mixtures contained, in a total volume of 1 mL, 80 mm Bicine (pH 8·6), 1·5 mm sodium pyrophosphate, 1 mm MgCl2, 0·4 mm NAD, 0·8 mm UDP glucose, 10 units of glucose-6-phosphate dehydrogenase (NAD-linked, from Leuconostoc mesenteroides), 4 units of phosphoglucomutase and 50 µL extract. The reaction was initiated with UDP glucose and was monitored spectrophotometrically at 340 nm.

Carbohydrate and total amino acids determination

About 0·1 g fresh leaf tissues were killed and incubated in 3 mL 90% ethanol at 60 °C for 2 h, homogenized and centrifuged. The pellet was re-suspended and extracted in 3 mL 90% ethanol two more times. The three resulting supernatants were combined and evaporated to dryness in a vacuum evaporator (Vacucenter; Lucerne, Switzerland). The residues were re-dissolved in 3 mL distilled water and centrifuged at 11 000 g for 15 min The clear supernatant was then used for non-structural carbohydrate and total amino acids determination. The remaining pellets following removal of soluble sugars were retained for starch analysis.

Sucrose content was determined as described by Farrar (1993). Starch content was determined by measuring glucose content after amyloglucosidase digest at 55 °C (pH 5·5) (Wang, Yuan & Quebedeaux 1997). Total amino acids were determined as described by Rosen (1957).

Total soluble protein and nitrogen content

Total soluble protein was determined with the dye binding method (Bradford 1976) using bovine serum albumin as standard. Total nitrogen was measured using the standard micro-Kjeldahl method.

Extraction and determination of plant hormones

The expanding leaves (leaf 1, 2 and 3 from the top) and aerial root tips were harvested at midday, frozen in liquid nitrogen immediately after fresh weight was taken, and stored at −80 °C until analysis. Extraction and immunoassay of indole-3-acetic acid (IAA) were carried out according to Chen et al. (1998). Extraction and immunoassay of gibberellin A1 & A3 (GA1+3) were carried out as described in Chen, Zhou & Zhang (1998). Extraction and immunoassay of isopentenyladenosine (iPA), zeatin riboside (ZR) and ABA were done as described in Chen et al. 1997).

Results

Growth and CAM activity

After 2 months of growth, the RGR of plants grown at elevated CO2 averaged 25% higher than that at ambient CO2 (Table 1). Shoot dry mass production per plant increased 31% while root dry mass production increased 98% under CO2 enrichment condition. There was also a 55% increase in root : shoot ratio in CO2-enriched plants.

Table 1.  RGR, shoot growth, aerial root growth, aerial root : shoot ratio and leaf total nitrogen in M. Yellow grown for 2 months at ambient and doubled-CO2. Different letters between columns indicate significant difference at P≤ 0·05 by Student’s t-test. Data for leaf total nitrogen were the means of samples harvested at 0800, 1200 and 1600 h local time. Samples for all the other parameters were harvested 1600 h local time. Data are means ± SE (n = 3–8)
 Ambient CO2Elevated CO2
RGR (g kg−1 d−1)6·80 ± 0·48b8·52 ± 0·78a
Shoot growth (g plant−1)0·32 ± 0·03b0·42 ± 0·05a
Aerial root growth
(mg plant−1)
61·7 ± 6·71b 122 ± 8·95a
Aerial root : shoot ratio0·22 ± 0·02b0·34 ± 0·03a
Leaf total nitrogen
(mg g−1 DW)
14·5 ± 1·45a12·6 ± 0·73a

Total titratable acidity of both plants with and without CO2 enrichment showed daily fluctuation of typical CAM plants (Fig. 1a). However, titratable acidity of samples collected at dawn and midnight from CO2-enriched plants was much higher than that from their ambient-CO2 grown counterparts. Similar patterns were found in malate accumulation (Fig. 1b).

Figure 1.

Total titratable acidity (a), malate content (b), nitrate content (c) and total amino acids content (d) in the leaves of M. Yellow grown at two CO2 concentrations. Vertical bars indicate SE (n = 4). Black bars on horizontal axes indicate night.

Enzyme activity

Initial Rubisco activity in M. Yellow increased in the light and reached maximum after 4 h in the light (Fig. 2a). Its activity declined gradually after 8 h exposure of light. Total Rubisco activity in M. Yellow also increased in the light but reached maximum after 8 h in the light and declined gradually thereafter (Fig. 2b). Both initial and total Rubisco activities in CO2-enriched plants were significantly lower than those in their ambient CO2-grown counterparts during the light period. However, this difference diminished gradually in the dark period, and eventually there was no significant difference in Rubisco activity between plants with and without CO2 enrichment. There was no significant difference in Rubisco activation state between plants with and without CO2 enrichment (Fig. 2c).

Figure 2.

Initial activity (a), total activity (b) and activation state (c) of Rubisco in the leaves of M. Yellow grown at two CO2 concentrations. Vertical bars indicate SE (n = 3). Black bars on horizontal axes indicate night.

SPS activity in M. Yellow increased in the light and reached maximum after 4 h of exposure to light (Fig. 3), after which SPS activity declined gradually. For samples taken at early morning, there was no significant difference in SPS activity between plants with and without CO2 enrichment. However, at all the other time points, SPS activity in plants grown at elevated CO2 was significantly higher than that at ambient CO2, especially in the saturating assay. After 4 h in the light, leaf SPS activity in plants grown at elevated CO2 was about 21 and 11% higher than that at ambient CO2 in the limiting and saturating assay, respectively. There was no significant difference in SPS activation state between plants with and without CO2 enrichment. SS activity in M. Yellow showed the same diurnal rhythm as SPS activity (Fig. 3d). SS activity in plants grown at elevated CO2 was markedly higher than that at ambient CO2 throughout the day. After 4 h in the light, SS activity in leaves of plants grown at elevated CO2 was about 18% higher than that at ambient CO2.

Figure 3.

Selective SPS activity (a), maximum SPS activity (b), SPS activation state (c) and SS activity (d) in the leaves of M. Yellow grown at two CO2 concentrations. Vertical bars indicate SE (n = 3). Black bars on horizontal axes indicate night.

During the daytime, PEPCase activity in plants grown at elevated CO2 was slightly lower than that at ambient CO2 (Fig. 4a). However, in the dark period, PEPCase activity in plants grown at elevated CO2 was markedly higher. At 6 h after light off, PEPCase activity in leaves of plants grown at elevated CO2 was nearly 40% higher than that at ambient CO2. Although leaf AGPase activity in CO2-enriched plants was slightly higher in the light period, there was no significant difference in leaf AGPase and UGPase activities between plants with and without CO2 enrichment (Figs 4c & d).

Figure 4.

PEPCase activity (a), AGPase activity (b) and UGPase activity (c) in the leaves of M. Yellow grown at two CO2 concentrations. Vertical bars indicate SE (n = 3). Black bars on horizontal axes indicate night.

Metabolite contents

There was no significant diurnal change in levels of nitrate, sucrose, and total soluble sugars in leaves of M. Yellow (Figs 1c, 5a & c). There was no significant difference in leaf nitrate, sucrose, and total soluble sugars content between plants with and without CO2 enrichment. However, leaf starch content increased following exposure to light and reached maximum at around 2 h after light off (Fig. 5b). Starch content in plants grown at elevated CO2 was significantly higher than that at ambient CO2 after 8 h exposure to light. At 2 h after light off, starch content in CO2-enriched plants was about 42% higher than that in their ambient CO2-grown counterparts. There was no significant diurnal change in total amino acids content in leaves of M. Yellow (Fig. 1d). However, total amino acids content in plants grown at elevated CO2 was markedly lower than that at ambient CO2 throughout the diurnal cycle.

Figure 5.

Sucrose content (a), starch content (b) and total soluble sugars content (c) in the leaves of M. Yellow grown at two CO2 concentrations. Vertical bars indicate SE (n = 4). Black bars on horizontal axes indicate night.

Levels of plant hormones

There was a remarkable increase in IAA, GA1+3, iPA and ZR content in expanding leaves of M. Yellow grown at elevated CO2, compared to their ambient CO2-grown counterparts (Table 2). However, in aerial root tips, only IAA and iPA content were significantly higher in high-CO2-grown plants. There was no significant difference in ABA content between plants with and without CO2 enrichment, in both expanding leaves and aerial root tips.

Table 2.  Levels of plant hormones in expanding leaves and aerial root tips of M. Yellow grown at ambient and elevated CO2 conditions. Samples were harvested at midday. Different letters indicate significant difference between CO2 treatments at P≤ 0·05 by Student’s t-test. Data are means ± SE (n= 4 different plants)
 Expanding leavesRoot tips
AmbientElevatedAmbientElevated
IAA content (nmol g−1 FW)0·30 ± 0·06b5·33 ± 1·88a 2·49 ± 0·34b31·34 ± 5·2a
GA1+3 content (nmol g−1 FW)3·81 ± 0·79b6·78 ± 0·62a 1·92 ± 0·47a 6·26 ± 3·40a
iPA content (nmol g−1 FW)0·32 ± 0·02b6·95 ± 0·61a 0·51 ± 0·07b0·768 ± 0·05a
ZR content (pmol g−1 FW)0·83 ± 0·28b2·64 ± 0·26a46·02 ± 2·25a49·68 ± 3·64a
ABA content (nmol g−1 FW)0·12 ± 0·02a0·19 ± 0·03a 0·19 ± 0·01a 0·28 ± 0·02a

Discussion

A 25% increase in RGR was observed in M. Yellow by a doubled atmospheric CO2 concentration. This is consistent with previous investigations on CAM species (Drennan & Nobel 2000). Whereas there was a 31% increase in shoot dry mass production, the dry mass production of aerial roots increased 98% leading to a 55% increase of aerial root : shoot ratio. Enhanced root growth in CAM and C3 species under CO2 enrichment has been reported previously (Drennan & Nobel 2000).

For the few CAM species that have been studied, carboxylation activity of Rubisco decreases but the activation state of Rubisco increases in response to a doubled CO2 concentration in midday-sampled leaves (Drennan & Nobel 2000). We also observed a much lower activity of Rubisco in plants grown at elevated CO2 during the daytime. However, we did not observe an overall difference in Rubisco activation state between plants with and without CO2 enrichment. Contrary to previous reports on CAM species Agave deserti and Opuntia ficus-indica, where decreased PEPCase activity in CO2-enriched plants was found in samples harvested at 2 h after dark (Israel & Nobel 1994; Graham & Nobel 1996), the activity of PEPCase in M. Yellow grown at elevated CO2 was much higher than that at ambient CO2 during the dark period. These results probably indicate an enhancement of dark CO2 fixation in these CO2-enriched M. Yellow plants.

Up-regulation of SPS under CO2 enrichment has been found in C3 species of rice (Seneweera et al. 1995; Hussain, Allen & Bowes 1999) and CAM species of Opuntia ficus-indica (Wang & Nobel 1996). In the present study, leaf SPS activity in M. Yellow grown at elevated CO2 was also up-regulated. Up-regulation of SPS could be achieved by three processes: coarse control through enhanced synthesis of SPS protein, or covalent modification of the protein via a phosphorylation and dephosphorylation cycle, and/or through fine control by metabolites acting as allosteric effectors (Stitt et al. 1988; Huber & Huber 1996). In this study, the SPS activity was assayed in vitro under the same substrate and effector concentrations. Thus, the higher SPS activity of CO2-enriched plants was not due to allosteric control. Neither was it due to protein phosphorylation because there was no significant difference in the activation state of SPS between plants with and without CO2 enrichment throughout the diurnal cycle (Fig. 4c). Therefore, the higher activity of SPS in CO2-enriched plants is most likely due to an increase in SPS protein. Further work is needed to verify this point.

Mokara Yellow plants grown at elevated CO2 showed a remarkable night-time increase in titratable acidity and malate accumulation (Fig. 1a & b). These results clearly indicate increased night-time CO2 fixation in these CO2-enriched plants. This is consistent with a much higher night-time PEPCase activity. In order to minimize the feedback inhibition on photosynthesis, the high-CO2-grown plants need enhanced capacity to metabolize the increased carbon gain. This is consistent with the present data, as both SPS and SS activities in CO2-enriched plants were significantly higher than those in plants without CO2 enrichment. SPS is a key control point in the sucrose formation pathway and the activity of SPS in leaf extracts appears to be related to the capacity of the leaf to form sucrose (Harbron, Foyer & Walker 1981; Amir & Preiss 1982; Huber & Huber 1996). SS-catalyzed metabolism has been linked with biosynthetic processes such as synthesis of starch (Winter & Huber 2000). The up-regulation of both SPS and SS activity might be an optimization process to facilitate the balance between increased carbon gain and its use. Interestingly, there was no significant difference in AGPase and UGPase activity between plants with and without CO2 enrichment, despite the fact that enhanced starch synthesis was found in CO2-enriched plants. These results indicate that AGPase activity is in excess in the leaves of M. Yellow plants. Indeed, SS activity, other than AGPase and starch synthase, was significantly correlated to the relative rate of starch synthesis in developing pea seeds (Déjardin et al. 1997). Using transgenic potato plants expressing SS antisense RNA, Zrenner et al. (1995) have shown that sucrose synthase is the major determinant of potato tuber sink strength. The data presented here also suggest that SS might play an important role in photosynthate partitioning in plants grown at elevated CO2 condition.

Despite an increase in sucrose formation in CO2-enriched plants, as evidenced by the up-regulation of SPS, leaf sucrose content and its daily change were similar under ambient and doubled CO2 concentration (Fig. 5a). This might be due to an increased sucrose export from these source leaves to sink organs such as the young leaves or roots. Indeed, sucrose transported into sink organs such as young daughter cladodes increased by doubling the atmospheric CO2 concentration in the CAM species of Opuntia ficus-indica (Wang & Nobel 1996). Maintenance of a constant sucrose level in the source leaves might prevent feedback inhibition of photosynthesis. The increase in leaf starch accumulation in CO2-enriched plants during the daytime does not limit its photosynthetic rate, as occurs for some C3 species as well as CAM species (Drennan & Nobel 2000).

With the exception of ABA, all plant hormones detected here showed a remarkable increase in the young leaves of M. Yellow grown at elevated CO2. This is interesting because ABA has been considered an inhibitor of elongation process in leaf growth (Zhang & Davies 1990). A study on effects of elevated CO2 and nitrogen nutrition on cytokinins in cotton found that cytokinin content increased only in low-nitrogen leaves under CO2 enrichment, whereas its content in leaf tissues was similar for high-nitrogen leaves grown at ambient and elevated CO2 (Yong et al. 2000). It is noteworthy that they only measured cytokinin content in the mature leaves (leaf 4 excluding cotyledons) of cotton. It is likely that the content of cytokinins in the young leaves of high-nitrogen cotton grown at elevated CO2 may also be higher than that grown at ambient CO2, as high-nitrogen cotton plants grown at elevated CO2 accumulated 80% more structural dry mass. In the present study, plants were watered daily with half strength Hoagland’s solution. It is unlikely that the plants were under nitrogen stress. This is supported by the fact that there was no significant difference in leaf total nitrogen content between plants with and without CO2 enrichment. Previous study on the distribution of plant hormones in aerial roots of CAM orchid showed that the highest levels of plant hormones were observed in root tips (Zhang et al. 1995). The significant increase of IAA and iPA content in root tips might be part of the reason for the enhanced aerial root growth of M. Yellow grown at elevated CO2. It has been proposed that auxin controls root cell elongation (Torrey 1976). It is widely accepted that root tips are the major sites of cytokinins biosynthesis. Cytokinins in the root play an important role in cell division and function of apical meristem (Torrey 1976; D’Agostino & Kieber 1999). It is interesting that there was no significant difference in GA1+3 content in root tips between M. Yellow plants with and without CO2 enrichment. There is little evidence supporting a role of gibberellins in root growth (Torrey 1976).

In conclusion, leaf SPS and SS activity in CO2-enriched M. Yellow increased significantly, along with a night-time increase in total titratable acidity and malate accumulation. Concomitantly there was a daytime down-regulation of both initial and total Rubisco activity in the leaves of CO2-enriched plants. These acclimation processes were beneficial because they enhanced both photosynthetic capacity at high CO2 and reduced resource investment in excessive Rubisco capacity. Although PEPCase activity was slightly lower in CO2-enriched M. Yellow during the daytime, it was much higher in the dark period in CO2-enriched plants than in their ambient CO2-grown counterparts. Although there was an increased leaf starch accumulation in the daytime in CO2-enriched plants, leaf sucrose content, total soluble sugars, total nitrogen and nitrate content were similar under ambient and elevated CO2 condition. The remarkable increase in the levels of IAA, GA1+3, iPA and ZR in the expanding leaves and root tips of plants grown at elevated CO2 might be one of the reasons for the enhanced growth of these plants.

Acknowledgments

We would like to thank Mr Whye Leng Chua for his assistance in the determination of total nitrogen content. We also thank Mr Tang Kwee Ong and Mr Wee Peng Yap for their skilled technical assistance. This research was supported by a grant from the National University of Singapore (R-154-000-065-112).

Received 22 August 2001;received in revised form 11 October 2001; accepted for publication 11 October 2001

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