INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant growth is initiated at the meristems and consists of several processes that include cell division, cell expansion, and differentiation (Taylor, 1997). Growth in elevated [CO₂] (EC) changes plant structure through its effects on primary and secondary meristems of shoots and roots (for review, see Pritchard et al., 1999). A selected review of literature (Ranasinghe and Taylor, 1996; Kinsman et al., 1997; Pritchard et al., 1999; Masle, 2000) indicated that cell division, cell expansion, and cell patterning of plants in EC may be altered by increased substrate (Suc) availability and possibly by differential expression of genes involved in cell cycling and cell expansion.

It has been established that plant hormones including cytokinins (CKs), abscisic acid, auxins, and gibberellins are involved in controlling developmental events within apical meristems such as cell division, cell elongation, and protein synthesis. Evidence to date supports the pivotal role of CKs in regulating plant cell division, differentiation, cyclin genes (for review, see D'Agostino and Kieber, 1999), and cell elongation (Rayle et al., 1982). In plant cell division CKs are required at three stages of the cycle: G₁/S transition, G₂/M transition, and cytoplasmic division (John et al., 1993; Zhang et al., 1996; Laureys et al., 1999; Riou-Khamlichi et al., 1999).

CKs are predominantly root-sourced plant hormones as it is widely accepted that root tips are the major sites of CK biosynthesis. CK translocation from the roots through the xylem to the aerial plant parts by the transpiration stream will control shoot development (Torrey, 1976; Letham and Palni, 1983; Letham, 1994). However, there are inconsistencies that weaken this hypothesis. There is evidence to show that meristematic plant tissues (other than root tips) are also capable of CK biosynthesis (Van Staden and Dimalla, 1981; Chen et al., 1985). If CKs are also synthesized in the shoots, root-sourced CKs may therefore be less important to leaf function. Nonetheless, it is more likely that root-sourced CKs play a greater role in mediating shoot growth in response to the conditions (e.g. root nutrition and temperature) in the root environment. Considering the enormous impact that growth in EC has on plant root systems (for review, see Rogers et al., 1996), it is possible that root CK production and supply through the xylem to the shoot may be altered in EC, and thereby may modify above ground growth (e.g. meristem size and leaf area) and developmental profile (e.g. apical dominance and branching).

In general, collection of bleeding xylem sap exuding from de-topped plants does give valuable information. However the composition of root-pressure bleeding sap is likely to be different from in vivo sap transported in an intact, transpiring plant, especially when bleeding sap is collected over an extended period. In addition, root-pressure sap is likely to have different solute content relative to sap in intact plants because phloem recirculation has ceased. The composition of the xylem sap is influenced by the return to the roots of solutes in the phloem (Schurr, 1998). An elegant approach that avoids this problem is the use of a root pressure chamber (Janes and Gee, 1973; Passioura, 1980; Munns and Passioura, 1984). The pneumatic pressure acting on the rooted soil is gradually increased and the plant water potential is elevated to an extent that the sap starts to exude from a small incision (e.g. leaf petiole) once the pressure balancing the tension created by transpiring leaves is reached. This technique has been employed by others seeking representative samples of the transpiration stream (Beck and Wagner, 1994; Else et al., 1995; Liang and Zhang, 1997; Jokhan et al., 1999).

The objective of this paper was to study the effects of EC on the growth of cotton (Gossypium hirsutum L. var Deltapine 90) plants and its associated levels of CKs in the xylem sap of leaf petioles and in leaf tissues. A better understanding of the effects of EC on CK levels and metabolism is important because growth changes in EC are probably initiated at the meristems where CKs have a crucial role in regulating cell proliferation. Special efforts were made to collect the xylem sap with minimal disruption to the whole plant transpiration. As nitrogen supply to the plant is also known to affect CK content (Samuelson et al., 1992; for review, see Jackson, 1993) and leaf growth (e.g. Sims et al., 1998b), the analysis was conducted on cotton plants growing at two levels of nitrogen nutrition in greenhouses under AC and EC.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Effects of [CO₂] and Nitrogren Nutrition on Whole Plant Growth and Photosynthesis

Cotton plants growing in 12 mM nitrate and under 720 ppm [CO₂] grew significantly more than those growing in AC (Table I). High-nitrogen plants accumulated 80% more structural dry mass (compare with 100% on a total dry mass basis) in EC, whereas those in low nitrogen treatments gained 60% increase in structural dry mass. High-nitrogen plants in EC produced 59% more leaf area than its control in AC, whereas low-nitrogen plants in EC gained 39% in leaf area. In particular, leaf 4, which was excised at the petiole for xylem sap collection (Fig. 1), of the high-nitrogen plants was 43% larger in terms of leaf area in EC, whereas leaf 4 growing in low-nitrogen was only 26% larger than the corresponding control plants in AC. EC also increased the root mass for high-nitrogen- (120%) and low-nitrogen-grown (90%) cotton plants. At both nitrogen treatments, shoot-to-root ratios (based on structural dry mass) of cotton plants grown in EC were generally lower than those grown in AC (Table I). Analysis of leaf tissue nitrogen showed that the low-nitrogen-grown plants in EC exhibited a significant reduction (23% on a structural dry mass basis; 20% on an area basis) in nitrogen content (Table II). In contrast, high-nitrogen-grown cotton plants in EC showed a reduction of 7% in their leaf nitrogen content when expressed on a structural dry matter basis. Leaf nitrogen content was similar when expressed on a leaf area basis for the high-nitrogen-grown cotton plants in AC and EC. The amount of TNC in leaves was highest for cotton leaves in EC and at low-nitrogen nutrition. Significant differences in specific leaf weight (expressed on a structural dry mass basis) were observed and this indicated that growth in EC resulted in more structural dry mass per unit leaf area for both nitrogen treatments (14% for low nitrogen; 7% for high nitrogen). At high nitrogen nutrition, photosynthetic rates per unit leaf area of cotton plants grown at AC and enriched [CO₂] were not significantly different when measured at a common [CO₂] (Fig. 2, a and b). However, there was a significant difference in low-nitrogen-grown plants. At low nitrogen nutrition, photosynthetic rates of plants grown in enriched [CO₂] were about 20% lower than plants grown in AC when measured at a common [CO₂].

View larger version (25K): [in this window] [in a new window]   Figure 1.   Simplified diagrammatic representation of the root pressure chamber with a cotton plant. The chamber was designed to use with a range of pot sizes and stem diameters. Sealing of the stem was achieved by using a conical shaped silicon rubber seal that is later compressed by a set of pressurizing plates. Note that pressure relief valves are not shown.

View this table: [in this window] [in a new window]   Table II.   Leaf nitrogen content, total non-structural carbohydrates (TNC) and specific leaf weight of cotton plants (31-36 d after emergence) Leaf nitrogen content (mg g1) and specific leaf weight (g m2) were calculated using structural dry mass. Data are means ± SE (n = 3-5).

View larger version (32K): [in this window] [in a new window]   Figure 2.   A, Light-saturated photosynthesis of leaves measured at 360 ppm [CO2] for low-nitrogen and high-nitrogen cotton plants growing in AC and EC; B, light-saturated photosynthesis of leaves measured at 720 ppm [CO2] for low-nitrogen and high-nitrogen cotton plants growing in AC and EC. Data were derived from three to five plants, means ± SE. All measurements were made in the greenhouse where the leaf temperature was 31°C to 35°C and vapor pressure difference between leaf and air was 1.2 to 1.8 kPa. The response of assimilation to intercellular [CO2] was measured at photosynthetic photon flux density of 1,400 µmol m2 s1 that was sufficient to saturate photosynthesis. White bars, AC- grown plants; hatched bars, EC-grown plants.

[CO₂] and Nitrogen Nutrition Effects on Xylem Sap CKs

We have used a simplified method for quantifying CKs in potentially active forms in the xylem sap and leaves. CK ribosides and nucleotides were converted to bases, whereas the O-, 7-, and 9-glucoside linkages were not cleaved appreciably. Thus throughout this paper, zeatin (Z), dihydrozeatin (DZ), N⁶-(2-isopentenyl) adenine (iP), Z-O-glucoside (OGZ), and DZ-O-glucoside (OGDZ) levels refer to the total content of each compound (base) in free, riboside, and nucleotide forms. Z, DZ, and iP (free and released) were detected in cotton xylem sap for all treatments (Table III). Z was the main CK base in the xylem sap and accounted for 90% to 96% of the total CK bases. The relative proportion of the three bases remained similar in nitrogen and [CO₂] treatments. EC increased the concentration of CKs in the sap at low nitrogen (144%) and high nitrogen (35%) treatments. Similarly, EC increased the delivery (on the basis of delivery rates, fmol s¹) of CKs to leaf 4 at low nitrogen (158%) and high nitrogen (70%) treatments. EC increased the delivery of CKs (delivery per unit leaf area, fmol m² s¹) from the roots to leaf 4 at low nitrogen (106%, on a leaf area basis) and high nitrogen treatments (17%, on a leaf area basis; Fig. 3). At AC, low-nitrogen leaves received 67% less CK delivery (per unit leaf area) than the high-nitrogen leaves, whereas low-nitrogen and high-nitrogen leaves have similar levels of CK delivery at EC. EC has no significant effect on the delivery per unit leaf area of CK O-glucosides from the roots to leaf 4 at low nitrogen and high nitrogen treatments (Fig. 4). OGDZ was the main O-glucoside detected in the sap and it was 2- to 7-fold more than the OGZ across all the treatments (data not shown). If we naively assume that all leaves received similar level of xylem-derived CKs as that of leaf 4, then the whole plant CK delivery per unit root dry mass can be estimated (Fig. 5). EC had little effect on the whole-plant CK delivery per unit root dry mass for low-nitrogen- and high-nitrogen-grown cotton plants. High-nitrogen-grown cotton plants had a 100% increase in the whole plant CK delivery per unit root dry mass over low-nitrogen-grown plants in AC and EC.

View this table: [in this window] [in a new window]   Table III.   Xylem sap flow rate, cytokinin concentrations (µmol m3), and delivery rates (fmol s1) of the sap collected at the petiole of leaf 4 of cotton plants (31-36 d after emergence) The value of each cytokinin base represented the total in free, riboside, and nucleotide forms. Data are means ± SE (n = 3-4). Cytokinin delivery rates to leaf 4 were calculated by multiplying the concentration by a sap flow rate. During xylem sap collection, sap flow rate was maintained at the level of transpiration (mmol s1) of the leaf prior to its excision. During the sap collection period, transpiration of an adjacent leaf (leaf 5) was continuously monitored to ensure that the transpirational flux of the whole plant was not significantly altered.

View larger version (46K): [in this window] [in a new window]   Figure 3.   Xylem sap CK delivery rate per unit leaf area (fmol m2 s1) in the petiole of cotton leaf 4. Data were derived from three to four cotton plants, means ± SE. CKs [zeatin, dihydrozeatin, and N6-(2-isopentenyl) adenine] were measured using scintillation proximity immunoassay. White bars, AC-grown plants; hatched bars, EC-grown plants.

View larger version (32K): [in this window] [in a new window]   Figure 4.   Xylem sap cytokinin O-glucoside delivery rate per unit leaf area (fmol m2 s1) in the petiole of cotton leaf 4. Data were derived from three to four cotton plants, means ± SE. CK O-glucosides (zeatin O-glucosides and dihydrozeatin O-glucosides) were measured using scintillation proximity immunoassay. White bars, AC-grown plants; hatched bars, EC-grown plants.

View larger version (39K): [in this window] [in a new window]   Figure 5.   Whole-plant CK delivery rate per unit root mass (fmol g1 s1) of cotton plants growing in AC and EC. The whole-plant CK [zeatin, dihydrozeatin, and N6-(2-isopentenyl) adenine] delivery per unit root dry mass was based on the assumption that all leaves received similar level of CKs from the roots as that of leaf 4. Data were derived from three to four cotton plants, means ± SE. CKs were measured using scintillation proximity immunoassay. White bars, AC-grown plants; hatched bars, EC-grown plants.

[CO₂] and Nitrogen Nutrition Effects on Leaf CK Content

Z, DZ, and iP (free and released) were detected in leaf tissues for all treatments (Table IV). The patterns of CKs in leaf tissues across treatments were also similar when expressed on either a dry mass basis or a structural dry mass basis (data not shown). The relative proportion of the three CK bases within each treatment changed in response to nitrogen and [CO₂] treatments. For example, high-nitrogen cotton leaves in EC contained proportionally less Z (39% of CK bases) than the high-nitrogen AC-grown cotton plants (65%). At AC, low- and high-nitrogen leaves had similar levels of Z (Table IV). In contrast, the high-nitrogen leaves in EC had a 500% decrease in Z levels (on a leaf area basis and a fresh mass basis). The levels for iP in leaves did not change appreciably for all treatments. There was a significant increase (164% on a leaf area basis; 126% on a fresh mass basis) of CKs in low-nitrogen leaves in EC when compared with the control plants in AC (Fig. 6, a and c). The CK content (per unit leaf area and per unit fresh mass) of high-nitrogen-grown plants in EC did not differ significantly from those growing in AC. Generally, EC has no effect on the CK O-glucoside content (per unit leaf area and per unit fresh mass) of leaf 4 in low and high nitrogen treatments (Fig. 6, b and d). OGDZ and OGZ were the two O-glucosides detected in the leaves and they remained in similar proportions across all the treatments (data not shown).

View this table: [in this window] [in a new window]   Table IV.   Leaf cytokinin content expressed on an area basis (nmol m2) and fresh mass basis (pmol g1) in leaf 4 of cotton plants (31-36 d after emergence) The value of each cytokinin base represented the total in free, riboside, and nucleotide forms. Data are means ± SE (n = 3-4).

View larger version (50K): [in this window] [in a new window]   Figure 6.   Leaf CK content in leaf 4 of cotton plants growing in AC and EC. A, Leaf CK [zeatin, dihydrozeatin, and N6-(2-isopentenyl) adenine] content expressed on a leaf area basis (nmol m2); B, leaf cytokinin O-glucoside (zeatin O-glucosides and dihydrozeatin O-glucosides) content expressed on a leaf area basis (nmol m2); C, leaf CK [zeatin, dihydrozeatin, and N6-(2-isopentenyl) adenine] content expressed on a fresh mass basis (pmol g1); D, leaf CK O-glucoside (zeatin O-glucosides and dihydrozeatin O-glucosides) content expressed on a fresh mass basis (pmol g1). Data were derived from three to four cotton plants, means ± SE. CKs were measured using scintillation proximity immunoassay. White bars, AC-grown plants; hatched bars, EC-grown plants.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The occurrence of a CK response to EC was investigated in cotton plants growing in two levels of nitrogen nutrition. Growth responses obtained in this experiment were similar to those found in previous experiments using a different cotton cultivar, Deltapine 16 (Wong, 1990). The increase in cotton plant growth in EC was generally attributed to the increase in the rate of photosynthesis per unit leaf area. Under these prevailing conditions, xylem sap and leaf tissues were obtained for CK analysis. During the collection of xylem sap, we have maintained the transpiration rate of the whole plant similar to that before the excision of the leaf. As the excised leaf was only about 8% to 14% of the total leaf area for the plant, a 10% error in the control of xylem sap flow at the cut end of the petiole would result in not more than 2% change in the transpiration rate of the whole plant grown at high nitrogen nutrition. This was because variations in stomatal conductance of various leaves of cotton plant grown at high nitrogen nutrition were small (data not shown). For low-nitrogen-grown cotton plants, the error can be slightly larger. Our estimate is that the error represented no more than a 4% change in the whole-plant transpiration rate.

Our ability to collect xylem sap with solutes identical to that entering the intact leaves coupled with the improved CK assay method for cotton xylem sap and tissues has provided new insights into CK metabolism. Thus the precise information on the level and identity of xylem-derived CKs entering a cotton leaf (Table III) coupled with the data on CK levels in this leaf (Table IV) enable xylem sap and foliar CKs to be compared concurrently for the first time. A similarity in relative levels of the different CK types might be expected between xylem sap and leaf, but was not observed (compare with Tables III and IV). Thus relative to Z (the predominant CK), DZ and iP were minor CKs in the xylem sap received by leaf 4. However, all three CK types were nearly equally prominent in the leaves (Table IV). This suggests that DZ and iP were conserved relative to Z in the leaves and/or that Z-type CKs may be converted to DZ. Conservation of the DZ-type CK can be accounted for by its stability to CK oxidase, which readily degrades Z. However, conservation of iP, which is degraded by CK oxidase and trans-hydroxylated to yield Z, is difficult to rationalize. The results emphasize the importance of CK metabolism in determining the CK status of leaves. Information of the type mentioned above derived from our xylem sap collection method should complement the commonly used approach to study CK metabolism based on radiolabeled compounds and excised organs.

[CO₂] and Nitrogen Nutrition Effects on Xylem Sap CKs

The CKs in the xylem sap of cotton plants grown in this experiment were predominantly (more than 90%) the trans-Z type. However, storage forms of CKs (OGZ and OGDZ) were also detected in the sap. Xylem sap collected from the petioles of high-nitrogen-grown cotton contained higher levels of CKs than those grown in low nitrogen in AC. This observation was also noted for stinging nettle (Urtica diocia L.) plants grown in two nitrogen nutritions (3 and 15 mM nitrate) and where xylem sap was collected from de-topped plants using a pressure chamber in AC (Beck and Wagner, 1994). An earlier study by the same research group using the same plant species and similar growth conditions reported no effect of nitrogen nutrition (3, 15, and 22 mM nitrate) on CKs collected from de-topped plants using natural root pressure (Fusseder et al., 1988). These examples highlight the importance of maintaining the normal whole-plant transpiration flux during sap collection.

Most of the previous studies of nitrogen effects on xylem sap CKs (Sattelmacher and Marschner, 1978; Salama and Wareing, 1979) were based on root pressure exudates collected after shoot excision. Usually, a low nitrogen supply resulted in a reduced CK level in the sap. These observations were confirmed by the present study with cotton plants grown in AC. We have established for the first time that increased nitrogen supply increases the actual delivery rate per unit leaf area of CKs to the leaf in AC. In EC, high-nitrogen- and low-nitrogen-grown leaves received similar levels (delivery rate per unit leaf area) of xylem CKs (Fig. 3). This observation implies that CK production was stimulated more in the low-nitrogen-grown cotton plants than in high-nitrogen-grown plants under EC. Furthermore, this could suggest that CK supply to the leaf is already physiologically optimized in high nitrogen nutrition conditions, and that doubling of [CO₂] only stimulates a slight increase in the CK delivery (Fig. 3). We cannot offer a physiological explanation for this phenomenon as the mechanism by which the nitrogen supply to the root is translated into a CK signal remains unclear (for review, see Stitt, 1999). Nonetheless, some progress was made in this area when a transcript for a CK-induced protein (pZmCip1) was isolated in maize leaves (Sakakibara et al., 1998). Sakakibara and coworkers (1998) showed that the addition of either nitrate or ammonium ions to the roots of intact maize plants led to an induction of phosphoenolpyruvate carboxylase (PEPc) in leaves, whereas the addition of nitrogen sources to the leaves did not. Induction of PEPc in the whole plant was associated with increased levels of CKs in the xylem sap and an increase of the transcript for the CK induced protein. Furthermore, the increase in PEPc and pZmCip1 transcripts could be mimicked by feeding exogenous CKs to detached leaves. Their results clearly implicated the synthesis of CKs in the roots in response to nitrogen ions. Nitrogen nutrition also markedly affects the fate of individual CK, which enters the cotton leaf. Thus supply of high nitrogen to EC grown plants did not appreciably change the Z delivery into leaf 4 (Fig. 3, where Z constituted 90% of the CK delivery rate per unit area), but did markedly lower (approximately 500%) the Z level in leaf 4 (Table IV). Hence, the rate of Z catabolism in the leaf appears to be greater in high-nitrogen- and EC-grown plants. The level of iP in leaf 4, unlike that of Z, changes little in response to growth [CO₂]. If CK metabolism is coupled to CK action as has been suggested, the stability of iP may result from a lack of involvement in mediation of [CO₂]-induced changes.

Our data (Figs. 3 and 6) can also be used to calculate an "apparent CK turnover time" in cotton leaves by dividing the leaf CK pool size (Fig. 6) by the CK delivery into the leaf (Fig. 3). The results for low-nitrogen plants are approximately 6 and 8 h at 360 and 720 ppm respectively, and for high-nitrogen plants, 3 and 2 h at 360 and 720 ppm respectively. Higher nitrogen nutrition appears to increase the CK turnover rate. However, this simplistic analysis still lacks several considerations such as the potential contribution of free CKs from CK O-glucosides, rate of glucosylation, diurnal effects on CK export from the roots, and biochemical interconversion between the different CKs. A detailed study to describe CK turnover in leaves will be presented later (J.W.H. Yong, unpublished data). At the whole-plant level, higher nitrogen nutrition increases the whole-plant CK delivery per unit root dry mass at AC and EC (Fig. 5). It is interesting that shoot exposure to either AC or EC appears to have little effect on the whole-plant CK delivery per unit root dry mass. This calculation was based on the assumption that all leaves received similar level of CKs as that of leaf 4. As a note of caution, expressing the whole-plant CK delivery per unit root dry mass basis may not be a good reflection of the physiological role of roots in relation to CK production as a large proportion of the root system consists of non-living structural tissues.

[CO₂] Effects on Leaf Growth by Altering CK Delivery to the Leaf

The remarkable outcome of studies with labeled CKs was the rapidity of their metabolism (Nooden and Letham, 1993). For example, ³H-ZR fed through the xylem was quickly metabolized to other compounds (approximately 80% within 1 h) and almost all (more than 95%) of the parent compound was metabolized within the 2nd h of incubation. Because of the rapid metabolism of xylem-derived CKs, there will be little or no accumulation of these CKs in active forms in the leaves. A decline in xylem-derived CKs should be rapidly sensed by the leaf. Hence, the root-leaf CK signal should be a very effective one. Our data indicated that leaf 4 received more xylem-derived CKs (delivery rate per unit area) in response to EC (Fig. 3). The magnitude of these differences in CK delivery between ambient [CO₂] (AC)- and EC-grown leaves was high in low-nitrogen conditions and lower in high-nitrogen conditions. There was, however, no direct and simple proportional link between the percentage increase in delivery of CKs arriving at the leaf in EC over AC, and the resultant increase in leaf area. For low-nitrogen-grown plants, we observed a 26% increase in area of leaf 4 at EC, and this was accompanied by a 106% (on an area basis) increase in CK delivery. On a whole-plant basis, low-nitrogen leaves were 14% "thicker" (greater structural dry mass per unit leaf area). In high-nitrogen plants, there was an increase of 43% in area of leaf 4 and a 17% (on an area basis) increase in CK delivery in response to EC. On the whole-plant basis, high-nitrogen leaves were 7% "thicker" (greater structural dry mass per unit leaf area).

The lack of a simple proportional link between leaf area and its CK fluxes in response to EC may reflect the complexity of CK metabolism and growth regulation at the cellular/molecular level. Also, part of the original xylem-derived CK signal arriving at the cotton leaf may be modified by foliar compartmentation within the leaf tissues. Sims and coworkers (1998b) encountered similar difficulties in their attempt to correlate leaf area and thickness with leaf expansion rate in EC. They suggested that EC probably had independent effects on leaf expansion rates and thickening of soybean leaves across a series of light and nitrogen gradients. Nonetheless, in the present study cotton leaves growing in two different nitrogen nutritions under EC received higher levels of CKs, although the function and fate of these xylem-derived CKs remained unclear. However, as discussed in the earlier section, a large proportion must have been catabolized. We also do not rule out the involvement of other hormones (e.g. gibberellins and abscisic acid) in mediating growth responses to EC.

Evidence drawn from cellular experiments further supports the role of CKs in mediating growth increases during exposure to EC, although none of these three studies mentioned here made direct measurements of CKs. Kinsman et al. (1997) provided evidence that exposure to EC stimulates primary growth of Dactylus glomerata shoots by increasing the proportion of rapidly dividing cells and shortening the cell cycle duration in shoot apices. The authors proposed that increased Suc availability in meristems during exposure to EC might have increased the proportion of rapidly dividing cells by stimulating cyclin activity. It is noteworthy that cyclins and cyclin-dependent kinases are proteins that regulate cell cycle and these proteins are under the influence of CKs (for review, see D'Agostino and Kieber, 1999). Ranasinghe and Taylor (1996) showed, using the primary leaves of Phaseolus vulgaris, that cell production and expansion were stimulated by EC. Increased leaf cell expansion is an important mechanism for enhanced leaf growth in EC, whereas the importance of increased leaf cell production in EC remains unknown. Similar observations were also made for the monocot, wheat (Masle, 2000). Masle observed a strong link between [CO₂] effects on cell division and expansion process in wheat. This link was substantiated by a good correlation between the spatial patterns of local rates of cell partitioning and elongation. A consistent effect of EC treatment was to reduce the time interval between successive cell divisions in expanding wheat leaves. It is therefore conceivable that the increased cell proliferation and cell expansion observed in P. vulgaris and wheat leaves, and the increase in primary growth of D. glomerata shoot apices, in EC was mediated by a greater delivery of CKs arriving at the shoot tissues from the roots. Since CO₂ is the substrate for photosynthesis, it is important to stress that the primary effect of EC on plant growth is the enhancement of photosynthesis.

All the above observations on increased cell division and cell expansion at EC can only occur when carbon and nitrogen are not limiting. As the xylem stream may only reach the transpiring plant parts and not the apical shoot tips and other major sites of cell division, the phloem may also be involved in the delivery of CKs at some stage in mediating growth regulation (Ziegler, 1975; Vonk, 1979; Komor et al., 1993; Lejeune et al., 1994). Future work that aims to resolve this question should involve xylem and phloem estimation of CK fluxes, cellular growth analysis (kinematic, anatomical, and anisotropy), and carbon translocation studies.

Is Photosynthetic Acclimation of Leaves at EC Influenced by the Levels of CKs?

Photosynthetic acclimation of leaves at EC is a complex and unresolved phenomenon (for review, see Sage, 1994; Drake et al., 1997; Stitt and Krapp, 1999), but predominantly modulated by sink limitation (Arp, 1991; Stitt, 1991), nitrogen limitation (Stitt and Krapp, 1999), and leaf age (Miller et al., 1997). It is widely accepted that the reduction of photosynthetic capacity in many plants grown in EC is attributed to the feedback effect of leaf carbohydrates on gene expression. However, recent data obtained by Sims and coworkers (1998a) who exposed soybean leaflets to a [CO₂] differing from that around the rest of the plant indicated that leaf carbohydrate may not be the crucial factor in reducing foliar photosynthetic capacity in EC. They proposed that mechanisms by which sink strength could alter leaf physiology and operate independently of changes in carbohydrate accumulation are likely to influence photosynthetic acclimation in EC. In his extensive review of the phenomenon of photosynthetic acclimation from a gas-exchange perspective, Sage (1994) speculated that the involvement of hormone-mediated signaling from roots at EC was a potential control point, which had remained largely unstudied. Our high-nitrogen-grown cotton leaves have similar levels of CKs in AC and EC. In contrast, there was a significant accumulation of CKs in the low-nitrogen leaves in EC. It is noteworthy that the levels of CKs in the low-nitrogen leaves in AC were similar to those of the high-nitrogen-grown cotton leaves in AC and EC. Similar levels of leaf CK content (per unit fresh mass) have been reported for mature leaves of stinging nettle growing in two different nitrogen nutritions (3 and 15 mM nitrate) at AC (Wagner and Beck, 1993). There was also a concomitant decrease in light-saturated photosynthesis of low-nitrogen cotton leaves growing in EC, when measured at 360 and 720 ppm [CO₂] in comparison with the control plants. In addition, these low-nitrogen leaves in EC have a high TNC content (Table II). With limitation to growth processes brought about by nitrogen, a "physiologically unbalanced" condition induced in EC may be manifested in part by the supra-optimal levels of active CKs in the leaves. We therefore postulate that these leaves under the prevailing low-nitrogen nutrition and EC environment were unable to fully utilize the xylem-derived CKs for physiological function. It is possible that an increase of active CK bases exceeded a threshold level that is unfavorable for photosynthetic activity in its broadest sense (e.g. NADH-dependent hydroxypyruvate reductase proteins are down-regulated by supra-optimal levels of iP, iP riboside, and ZR [Wingler et al., 1998]).

Studies with transgenic plants expressing a CK biosynthesis gene also show that photosynthetic acclimation of leaves to [CO₂] could be associated with hormonal imbalance. These studies could be interpreted in terms of threshold levels for CKs in leaves, which when exceeded can lead to pronounced physiological effects (for review, see Synková et al., 1997). Generally, a small increase in endogenous CKs is associated with slight, often positive effects on photosynthetic characteristics (e.g. atský et al., 1993). At high CK levels, most of the foliar photosynthetic activities are smaller. If we assume that the high-nitrogen-grown cotton leaves in AC and EC maintained optimal levels of CKs in the leaves for physiological function, it is possible that the low-nitrogen leaves in EC exceeded the optimal levels of CKs. This may lead to a decline in Rubisco (the major plant protein) content that eventually causes a decline in leaf nitrogen content and photosynthesis (Table II; Fig. 2). This view is supported by the observation that gene promoter activity of the small subunit of Rubisco was reduced by supra-optimal levels of CKs (Gaudino and Pikaard, 1997). Of course it is also possible that CKs accumulated because they could not be utilized and that foliar photosynthetic capacity was lower for some unrelated reason. Clearly more work needs to be done to clarify the involvement of CKs, and possibly the other plant hormones in regulating photosynthetic acclimation at EC.

In conclusion, the data presented here demonstrated that higher nitrogen nutrition increased the delivery (per unit leaf area) of CKs from the roots through the xylem to the cotton leaf at AC. EC caused a greater increase in CK delivery to the cotton leaf at low nitrogen conditions than at high nitrogen conditions. CK content (per unit leaf area and per unit fresh mass) in leaf tissues was similar for high-nitrogen leaves growing in AC and EC. A significant increase in CK content in low-nitrogen leaves was induced by EC. The increased levels of xylem-derived CKs arriving at the leaf tissues growing in EC are likely to provide the necessary hormonal cues to alter leaf growth and development at the cellular/molecular level. The ability of leaf tissue to utilize the increased delivery of CKs in EC may be an important determinant in understanding photosynthetic acclimation of leaves in EC.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Growth Conditions

Seeds of cotton (Gossypium hirsutum L. var Deltapine 90) were sown in 4.5-L polyvinyl chloride pots containing sterilized soil mixture (3 parts sand:1 part loam). Uniform seedlings were selected during thinning from six to one per pot after germination. Experiments were carried out on 31- to 36-d-old plants with seven to 10 leaves. The two adjacent greenhouses were well-ventilated and matched for temperature (30°C ± 2°C day, 20°C ± 2°C night) and relative humidity (45% ± 10% day, 70% ± 10% night). The [CO₂] of one greenhouse (ambient) was run at 360 ± 15 ppm, whereas the other greenhouse was run at an EC of 720 ± 30 ppm. The plants were grown under full sunlight between late summer and early autumn, the midday irradiance being 1,600 µmol m² s¹. The daylength decreased slowly over the duration of the experiment (February through April 1999) from 13 to 11 h according to the seasonal variation existing in Canberra, Australia. The potting mixture in each pot was flushed daily in the early morning with 1 L of Hewitt's nitrate nutrient (Hewitt and Smith, 1975), consisting of 4 mM K⁺, 4 mM Ca²⁺, 1.5 mM Mg²⁺, and 1.33 mM H₂PO₄ with balancing SO₄² and Cl anions and micronutrients. There were two nitrate concentrations: 12 and 2 mM. Plants were watered in the late afternoon to compensate for the water loss due to transpiration.

Leaf Gas Exchange Measurements

Leaf gas exchange was measured using an open system gas exchange apparatus (LI-6400, LI-COR, Lincoln, NE) equipped with the standard leaf chamber, light-emitting diode light source, and the [CO₂] injector system for control of [CO₂]. The response of assimilation to intercellular [CO₂] was measured at photosynthetic photon flux density of 1,400 µmol m² s¹ that was sufficient to saturate photosynthesis. All measurements were made in the greenhouse where the leaf temperature was 31°C to 35°C and vapor pressure difference between leaf and air was 1.2 and 1.8 kPa.

Xylem Sap Collection

We designed and built a root pressure chamber with a split lid to house a polyvinyl chloride pot (maximum dimensions of a 160-mm diameter by a 600-mm length; Fig. 1). The xylem water potential of a cotton plant whose roots are enclosed in the chamber can be altered by varying pneumatic pressure. The through flow rate of the pressurizing gas was about 1 L per min. The partial pressure of O₂ of the pressurizing gas was maintained at 21 kPa by mixing O₂ with N₂. This was achieved by using computer-controlled motorized needle valves. Each sap collection exercise took place on a cloudless day within the greenhouses (360 or 720 ppm CO₂) where the plants were growing, to minimize changes to the whole-plant transpiration rates.

Transpiration rate of the youngest fully expanded leaf (usually leaf 4, counting after the cotyledons) was calculated using the transpiration rate per unit leaf area and the leaf area. After sealing the pot into the pressure chamber, the leaf 4 was excised. The pneumatic pressure was raised gradually until it reached slightly above zero water potential such that the rate of sap collection was equal to the transpiration rate, before excision, of leaf 4. Xylem sap flowing out of the petiole was monitored by measuring the speed of the sap flowing through small-diameter polyethylene tubing.

We also monitored the transpiration rate and the other gas exchange parameters of an adjacent leaf (leaf 5, slightly younger than leaf 4) before excision of the target leaf and throughout the sap collection period. This was done to ensure that there was no significant change to the whole-plant transpiration rate during the sap collection period.

Preliminary studies have shown that leaf 5 had similar gas exchange characteristics to leaf 4. The removal of leaf 4 only resulted in the loss of 8% to 14% of the total leaf area. The first approximately 200 µL or more of sap was discarded to avoid contamination by cut cells and each sap collection exercise took about 2 to 4 h (between 7 AM and 12 PM) to minimize distortion to sap flow and wound-induced contamination (Else et al., 1994; Jokhan et al., 1999). A possible, but unlikely, contamination by phloem sap was checked by analyzing for Suc. The results were negligible. Sap for solute analysis was collected in glass vials (kept at 0°C in an ice bath) throughout the collection period. Immediately after collection, sap samples were frozen and stored at 20°C. Leaf area and fresh weight were measured immediately after excision and the leaf was wrapped in aluminum foil and immersed in liquid nitrogen. Leaf area was measured with a leaf area meter (LI-3000, LI-COR). Leaf tissues were later stored in 80°C until further analysis. After collecting xylem sap, shoots and roots of the plants were harvested for leaf area and mass (fresh and dry) determination. Oven-dried (80°C) tissues were used for nitrogen and TNC analyses.

CK Analysis

We used a simplified method for quantifying CKs in potentially active forms in the xylem sap and leaves. This method was based on the conversion of 9-ribosides and nucleotides to bases, purification of the total bases using HPLC, and their quantification by scintillation proximity immunoassay (Wang et al., 1995). Some modifications to this method were made for processing cotton xylem sap and leaf tissues and these will be described in the following section. During this conversion, only the 9-ribosides and nucleotides were converted to bases, whereas the O-, 7-, and 9-glucoside linkages were not cleaved appreciably.

Leaf Extract Preparation and Treatment

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Chromatographic Fractionation of Leaf Extracts Prior to Scintillation Proximity Assay (SPA)

Xylem Sap Preparation, Treatment, and Purification

HPLC

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Digestion and Chromatographic Fractionation of O-Glucosides Analysis Prior to SPA

SPA

Tissue Analysis of Nitrogen and TNC

Leaf nitrogen content was measured using an elemental analyzer (EA 1110, CE Instruments, Italy) that was periodically calibrated with glucosamine standards. The TNC in leaf and stem tissues were determined according to Wong (1990) and used for calculating structural dry matter content.