Planta
Planta (1988)173 : 352-366
9 Springer-Verlag 1988
Growth response of barley and tomato to nitrogen stress and its control by abscisic acid, water relations and photosynthesis F. Stuart Chapin, III 1, Colin H.S. Walter 2 and David T. Clarkson 2 1 Institute of Arctic Biology, University of Alaska, Fairbanks, A K 99775, USA 2 Department of Agricultural Sciences, University of Bristol, Long Ashton Research Station, Long Ashton, Bristol BS18 9AF, U K
Abstract. Barley (Hordeum vulgare L.) and tomato Lycopersicon esculentum Mill.) were grown hydroponically and examined 2, 5, and 10 d after being deprived of nitrogen (N) supply. Leaf elongation rate declined in both species in response to N stress before there was any reduction in rate of dryweight accumulation. Changes in water transport to the shoot could not explain reduced leaf elongation in tomato because leaf water content and water potential were unaffected by N stress at the time leaf elongation began to decline. Tomato maintained its shoot water status in N-stressed plants, despite reduced water absorption per gram root, because the decline in root hydraulic conductance with N stress was matched by a decline in stomatal conductance. In barley the decline in leaf elongation coincided with a small (8%) decline in water content per unit area of young leaves; this decline occurred because root hydraulic conductance was reduced more strongly by N stress than was stomatal conductance. Nitrogen stress caused a rapid decline in tissue NO~- pools and in N O ~ flux to the xylem, particularly in tomato which had smaller tissue NO;- reserves. Even in barley, tissue N O ~ reserves were too small and were mobilized too slowly (60% in 2 d) to support maximal growth for more than a few hours. Organic N mobilized from old leaves provided an additional N source to support continued growth of N-stressed plants. Abscisic acid (ABA) levels increased in leaves of both species within 2 d in response to N stress. Addition of ABA to roots caused an increase in volume of xylem exudate but had no effect upon N O 3 flux to the xylem. After leaf-elongation rate had been reduced by N stress, photosynthesis declined in both barley and tomato. This decline was associated with increased leaf A B A A bbreviation and symbols: ABA = abscisic acid; ci = leaf internal CO2 concentration; Lp = root hydraulic conductance
content, reduced stomatal conductance and a decrease in organic N content. We suggest that N stress reduces growth by several mechanisms operating on different time scales: (1) increased leaf ABA content causing reduced cell-wall extensibility and leaf elongation and (2) a more gradual decline in photosynthesis caused by ABA-induced stomatal closure and by a decrease in leaf organic N.
Key words: Abscisic acid and nitrogen stress - Hordeum (ABA, N stress) Hydraulic conductance Lycopersieon (ABA, N stress) - Nitrate (storage, transport) - Nitrogen stress and growth - Photosynthesis and N stress.
Introduction Although nitrogen (N) supply usually limits growth of both crops and wild species under field conditions, the precise mechanisms by which this occurs are unclear. Limited supply of N reduces rates of cell division, cell expansion, photosynthesis, leaf production and tillering (for reviews, see Clarkson and Hanson 1980; Chapin 1980), yet it is not known which of these processes is affected first or most strongly by N deficit. In general, N deficit reduces leaf area development more strongly than it reduces photosynthetic rate (Watson 1952). Radin and colleagues (Radin and Boyer 1982; Radin and Eidenbock 1984) suggest that the decline in leaf-elongation rate (and therefore leafarea development) is a consequence of reduced water transport to the shoot which reduces shoot water potential and turgor for cell enlargement. Thus, the initial effect of N stress upon growth may act through a change in water relations rather than through its effect on photosynthesis. Drought
F.S. Chapin III et al.: Mechanisms of growth response to nitrogen stress
and saline conditions also reduce leaf elongation before photosynthesis is affected (Wardlaw 1969; Munns et al. 1982; Rawson and Munns 1984), and it has been generally assumed that this is primarily a consequence of reduced turgor in expanding cells (e.g. Boyer 1968; Hsiao 1973). However, several recent studies have shown that drought or salt stress reduces leaf elongation even when shoots are kept at full turgor, perhaps due to some signal from the roots (e.g. lack of cytokinins) that directly or indirectly reduces cell-wall extensibility (Matsuda and Riazi 1981; Michelena and Boyer 1982; Matthews et al. 1984; Blackman and Davies 1985; Termaat et al. 1985; Schulze 1986a). Flooding also stops leaf elongation before turgor is affected (Wadman-van Schravendijk and van Andel 1985). A similar change in root signal, rather than changes in leaf turgor, might explain the early reduction in leaf elongation in response to N stress. The present study was undertaken to consider several possible mechanisms by which N stress might reduce growth. In barley and tomato, we examined changes in photosynthesis, water relations, abscisic acid (ABA; a hormone implicated in both N and water stress), and N mobilization from internal stores. Material and methods Seedlings of barley (Hordeum vulgare L. cv. Midas) and tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) were grown in solution culture which supplied all N as NO~- until two leaves had reached full expansion, as described by Chapin et al. (1987). Then, at the start of the experiment (day 0), half of the plants were maintained in complete nutrient solution containing 10 m M NO~-, and half were transferred to a solution of similar osmolarity lacking NO~-. Measurements were conducted at days 0, 2, 5 and 10. All nutrient solutions were replenished at days 0 and 5. At each harvest date, barley plants were subdivided into roots, old leaves (i.e. leaves 1 and 2) and young leaves (the remainder of the shoot). Tomatoes were subdivided into roots and shoots. Dry weights were determined after oven-drying for 48 h at 60 ~ C. On some plants, old and young leaves were subsampled into liquid nitrogen for whole-tissue ABA analysis, as described elsewhere (Chapin et al. 1987). In tomato, old leaves consisted of the leaflets of the two oldest true leaves; young leaves were the leaflets of the two largest expanding leaves. Length of the lamina (barley) or terminal leaflet (tomato) was measured periodically on selected leaves. Root sugar concentrations were measured on whole root axes of barley and tomato plants by boiling in 25 or 50 ml 80% ethanol for 10 rain, then decanting the supernatant. The root was treated with two further volumes of boiling 80% ethanol. The washings were combined with the original extract and, when cool, made up to 50 or 100 ml with 80% methanol. A subsample of 7 ml was evaporated to dryness under vacuum and redissolved in either 2 or 4 ml water. Total sugars were determined by autoanalyzer using a slightly modified version
353
of the sulfuric acid-orcinol method (Shannon 1972); reducing sugars were determined by the copper-bicinchoniate procedure (Mopper and Gindler 1983).
Tissue water content per unit leaf area was calculated from fresh:dry weight subsamples used in A B A analyses (Chapin et al. 1987) and from specific leaf weight (see below). Dried samples were then ground in a Wiley mill. One subsample (0.1 g) was heated in 5 ml distilled water at 90 ~ C for 2 h (mixed thoroughly every 0.5 h), filtered and analyzed for NO~- concentration (Brewer et al. 1966); a second subsample (0.4 g) was digested in selenous-sulfuric acid and analyzed for organic N by micro Kjeldahl using the salicylate-nitroprusside method. Data are expressed on a fresh-weight basis. In vivo nitrate-reductase activity was determined in freshly excised new and old leaves following vacuum infiltration. Samples were incubated in the dark for 1 h at 20 ~ C in 1.5% propan-l-ol containing 50 m M NO~ and 50 m M phosphate, pH 7.0 (Stewart et al. 1973). Leaves were then boiled for 10 min, and N O r concentration was measured on these extracts and on subsamples collected before incubation (Brewer et al. 1966). Leaf water potential was measured by inserting leaves into a pressure chamber. Between times of collection and measurement, leaves were kept in the dark in a plastic bag with wet filter paper. Water absorption rate was measured for intact tomato plants from the decrease in volume of solution culture over 2-h intervals (as compared with volume changes in an aerated solution containing no plant). Photosynthesis and transpiration were measured on intact leaves with a closed-system portable infrared gas analyzer (model 6000; Licor, Lincoln, Neb., USA), fitted with a Vaisala humidity sensor. From these measurements stomatal conductance and leaf internal CO2 concentration were calculated. Each measurement was initiated as soon as the rate of COz depletion in the chamber stabilized, generally within 30 s. Depletion of CO2 during each measurement averaged 20 ppm. All measurements were made at the photon fluence rate (300 lamol.m-Z.s -1, 40(~700 nm), temperature (20 ~ C), and humidity (50%) at which plants were grown in order to estimate photosynthesis under ambient growth conditions. Flow rate was adjusted to maintain constant relative humidity during the measurement period. The area of leaf samples from photosynthetic measurements was measured on a leaf-area meter (Delta T Devices, Burwell, Cambridge, U K ) ; then leaves were dried and weighed to determine specific leaf weight. Fluxes of water (J~), NO~ and total solutes into the xylem under normal growing conditions were estimated for excised root systems by placing them in the solutions in which plants were grown (i.e. roots of N-sufficient plants in complete nutrient solution and roots of N-stressed plants in nutrient solutions of similar osmolarity but lacking N O ~ ) and collecting and analyzing the xylem exudate (Chapin et al. 1981). Osmolarity of the xylem sap and external solution was measured and used to calculate root hydraulic conductance (Lv), as described by Glinka (1980) and Radin and Boyer (1982). The effect of exogenous ABA upon these transport parameters was determined, as described elsewhere (Chapin et al. 1987).
Results
Growth. Relative growth rate (RGR) of N-sufficient controls was higher in tomato (0.21 g . g - a .
354
F.S. Chapin III et al. : Mechanisms of growth response to nitrogen stress
TOMATO
BARLEY 6000
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0 L i 2
J .
i
i
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4
6
8
(h)
Fig. 6. Nitrate flux o f xylem, N O 3 , concentration, and volume o f xylem exudate in excised r o o t s o f barley and t o m a t o exposed to the solutions in which they were grown. D a t a are means 4-SE, n = 4. Statistics as in Fig. 1
T a b l e 2. Effect o f N stress on osmolarity o f xylem sap and hydraulic conductance o f excised roots. D a t a are means _+ SE, n = 4.
Statistics as in Fig. I Species
Barley Tomato
Duration of N stress (d)
5 2 4
Osmolarity (mmol-1 1)
Hydraulic conductance ( p l . g - 1 . h -1 . k P a - 1 )
+N
--N
+N
--N
88.0 _+ 1.7 39.5 _+ 1.9 28.5_+0.5
82.7-t-0.9" 30.8_+1.4" 27,8_+0.3
0.20_+0.01 4.2 _+0.7 8.1 _+1.3
0.13_+0.01 * 1.4 _+0.2* 1.4 _+0.0"
and tomato roots (caused by the small root pool available for transport) and (2) a smaller exudate volume (J~) in tomato immediately following excision. The smaller J~ in tomato was apparently the result of a lower hydraulic conductance (Lp, Table 2). Jv increased during the 6 h after excision in N-stressed roots, partially compensating for the difference in NO 3 flux between N-stressed roots and controls. The differential between initial NO;fluxes in N-stressed and control roots became more pronounced with time (day 4 or 5 versus day 2) and was greater in tomato (Fig. 6) which had the lowest concentration of root NO~ reserves (Fig. 4).
Water-absorption rates of entire plants (groot; Table 1) were 10-fold higher than J~ in excised roots (Fig. 6), indicating that root pressure accounts for only a small proportion of the water transport in the intact plant. Similarly, the rate of organic-N accumulation in the shoot (calculated from Fig. 5) was threefold higher than that observed for the NO~- flux to the xylem of excised roots (Fig. 6), indicating that the transpiration stream enhances NO 3 transport to the shoot. Exogenous ABA generally caused no significant change in NO 3 flux to the xylem, because increases in J~ (in barley N-stressed roots and in tomato N-stressed roots at day 2) tended to dilute
358
F.S. Chapin III et al. : Mechanisms of growth response to nitrogen stress
Table 3. Effect of exogenous ABA on NO 3 flux to xylem, NO 3 concentration, and volume of xylem exudate in excised roots of barley and tomato after varying duration of N stress. " T " indicates 0.1 gM 2-trans-ABA (the biologically inactive isomer); otherwise ABA (the biologically active isomer) was applied at the indicated concentrations (~tM). Data are means _+SE, n =4. Statistics indicate significant differences from 0 ABA addition, as described in Fig. 1 [AB~
0
T
0.01
0.1
1.0
0.53 +0.12 0.14 _+0.04
0.65 +0.15 0.17 +_0.01
0.67 _+0.17 0.16 +0.02
0.50 - 0 . 1 0 0.020 _+0.002
0.73 +_0.13 0.023 +_0.002
0.70 +_0.12 0.028 -+ 0.004
NO 3 flux to xylem ( g m o l . g - 1. h - 1) Barley Day 2 +1 --N
0.41 • 0.04 0.11 _+0.01
Day 5 +N --N
0.68 _+0.08 0.022 -+ 0.004
0.52 _+0.06 0.016_+0.005
Day 2 +N -N
0.84 _+0.08 0.051 _+0.002
0.72 _+0.04 0.051 -+0.003
1.13 +-0.28 0.074 _+0.007 *
Day 4 +N -N
0.65 _+0.08 0.018_+0.001
0.69 -+0.16 0.016 -+ 0.001
0.85 _+0.08 0.028 _+0.005
Tomato
Xylem NO3 concentration (mmol.1 1) Barley Day 2 +N --N
25.4 +_ 1.4 2.6 _+0.3
Day 5 +N --N
21.8 _+ 1.2 0.90+_0.13
21.4 _+1.0 0.65_+0.21
19.2 _+ 1.2 1.07+_0.15
18.4 -+ 1.5 1.11 _+0.02
18.8 -+1.3 1.11-+0.10
13.1 _+0.7 0.19_+0.04
12.8 _+0.5 0.14_+0.003
13.2 +-0.6 0.22_+0.05
25.7 _+0.3 2.5 _+0.5
23.6 -+2.1 2.5 _+0.1
25.7 + 1.3 2.3 +0.1
21.5 • 0.74 _+0.08
25.0 +-2.3 0.65-+0.08
23.1 • 1.2 0.75_+0.11
Tomato Day 2 +N -N Day 4 +N --N
Exudate volume (gl. g - 1.h t) Barley Day 2 +N -N
23_+3 34-+3
Day 5 +N --N
33_+3 24_+ 1
26+_1 25+_0
Day 2 +N --N
136+_17 81_+7
97_+6 77_+5
201 _+27 115_+3"
Day 4 +N --N
87-+9 81_+9
99_+19 91_+4
113+8 98_+10
27_+5 51+_3"
27_+5 72_+4**
21_+5 72_+4**
26_+6 28_+2
33+_8 36+_2*
33_+7 37+ *
Tomato
F.S. Chapin III et al. : Mechanisms of growth response to nitrogen stress
359
BARLEY .
TOMATO .
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Fig. 7. Photosynthesis, stomatal conductance and internal CO2 concentration of barley and tomato leaves of plants grown with and without NO~-. Data are means + S E , n - 1 0 . Statistics as in Fig. 1
exudate N O 3 concentration to give a nearly constant NO~- flux (Table 3). Nitrogen-sufficient roots did not respond to exogenous A B A in either species. The biologically inactive 2-trans-ABA had no significant effect upon any measured parameter.
Gas exchange. Young barley leaves had higher photosynthetic rates than old leaves; in both young and old leaves rates declined with time (Fig. 7). Photosynthetic rate was increased by N stress in young leaves at day 2, reduced by N stress in old leaves by day 5 and reduced by N stress in all leaves by day 10. Stomatal conductance roughly paralleled changes in photosynthetic rate, declining with leaf age, increasing with N stress in young leaves at days 2 and 5, declining with N stress in old leaves at day 5 and declining with N stress in young leaves at day 10. Because of the parallel changes in photosynthesis and stoma-
tal conductance with N stress, leaf internal CO2 concentration (ci) was not affected by N stress (Fig. 7). Specific leaf weight declined only slightly with N stress (Fig. 3), so that patterns of photosynthesis expressed per unit tissue weight were similar to those shown in Fig. 7. Photosynthetic rate was closely correlated with both organic-N content (r = 0.68 **) and stomatal conductance ( r = 0.82**), indicating that the latter was closely adjusted to the changing photosynthetic potential of the leaf. Consequently, ci was not affected by N stress (Fig. 7). Photosynthetic rate in N-sufficient tomatoes was nearly twice that measured in barley, and, unlike barley, showed no consistent change with leaf number or leaf age (Fig. 7). In tomato, photosynthetic rate decreased in response to N stress in old leaves by day 2 and in all leaves by day 5. Stomatal conductance was not closely coupled to photosynthetic rate in tomato, in that it declined with age
360
F.S. C h a p i n III et al. : M e c h a n i s m s o f growth response to nitrogen stress
i
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Planta (1988)173 : 352-366
9 Springer-Verlag 1988
Growth response of barley and tomato to nitrogen stress and its control by abscisic acid, water relations and photosynthesis F. Stuart Chapin, III 1, Colin H.S. Walter 2 and David T. Clarkson 2 1 Institute of Arctic Biology, University of Alaska, Fairbanks, A K 99775, USA 2 Department of Agricultural Sciences, University of Bristol, Long Ashton Research Station, Long Ashton, Bristol BS18 9AF, U K
Abstract. Barley (Hordeum vulgare L.) and tomato Lycopersicon esculentum Mill.) were grown hydroponically and examined 2, 5, and 10 d after being deprived of nitrogen (N) supply. Leaf elongation rate declined in both species in response to N stress before there was any reduction in rate of dryweight accumulation. Changes in water transport to the shoot could not explain reduced leaf elongation in tomato because leaf water content and water potential were unaffected by N stress at the time leaf elongation began to decline. Tomato maintained its shoot water status in N-stressed plants, despite reduced water absorption per gram root, because the decline in root hydraulic conductance with N stress was matched by a decline in stomatal conductance. In barley the decline in leaf elongation coincided with a small (8%) decline in water content per unit area of young leaves; this decline occurred because root hydraulic conductance was reduced more strongly by N stress than was stomatal conductance. Nitrogen stress caused a rapid decline in tissue NO~- pools and in N O ~ flux to the xylem, particularly in tomato which had smaller tissue NO;- reserves. Even in barley, tissue N O ~ reserves were too small and were mobilized too slowly (60% in 2 d) to support maximal growth for more than a few hours. Organic N mobilized from old leaves provided an additional N source to support continued growth of N-stressed plants. Abscisic acid (ABA) levels increased in leaves of both species within 2 d in response to N stress. Addition of ABA to roots caused an increase in volume of xylem exudate but had no effect upon N O 3 flux to the xylem. After leaf-elongation rate had been reduced by N stress, photosynthesis declined in both barley and tomato. This decline was associated with increased leaf A B A A bbreviation and symbols: ABA = abscisic acid; ci = leaf internal CO2 concentration; Lp = root hydraulic conductance
content, reduced stomatal conductance and a decrease in organic N content. We suggest that N stress reduces growth by several mechanisms operating on different time scales: (1) increased leaf ABA content causing reduced cell-wall extensibility and leaf elongation and (2) a more gradual decline in photosynthesis caused by ABA-induced stomatal closure and by a decrease in leaf organic N.
Key words: Abscisic acid and nitrogen stress - Hordeum (ABA, N stress) Hydraulic conductance Lycopersieon (ABA, N stress) - Nitrate (storage, transport) - Nitrogen stress and growth - Photosynthesis and N stress.
Introduction Although nitrogen (N) supply usually limits growth of both crops and wild species under field conditions, the precise mechanisms by which this occurs are unclear. Limited supply of N reduces rates of cell division, cell expansion, photosynthesis, leaf production and tillering (for reviews, see Clarkson and Hanson 1980; Chapin 1980), yet it is not known which of these processes is affected first or most strongly by N deficit. In general, N deficit reduces leaf area development more strongly than it reduces photosynthetic rate (Watson 1952). Radin and colleagues (Radin and Boyer 1982; Radin and Eidenbock 1984) suggest that the decline in leaf-elongation rate (and therefore leafarea development) is a consequence of reduced water transport to the shoot which reduces shoot water potential and turgor for cell enlargement. Thus, the initial effect of N stress upon growth may act through a change in water relations rather than through its effect on photosynthesis. Drought
F.S. Chapin III et al.: Mechanisms of growth response to nitrogen stress
and saline conditions also reduce leaf elongation before photosynthesis is affected (Wardlaw 1969; Munns et al. 1982; Rawson and Munns 1984), and it has been generally assumed that this is primarily a consequence of reduced turgor in expanding cells (e.g. Boyer 1968; Hsiao 1973). However, several recent studies have shown that drought or salt stress reduces leaf elongation even when shoots are kept at full turgor, perhaps due to some signal from the roots (e.g. lack of cytokinins) that directly or indirectly reduces cell-wall extensibility (Matsuda and Riazi 1981; Michelena and Boyer 1982; Matthews et al. 1984; Blackman and Davies 1985; Termaat et al. 1985; Schulze 1986a). Flooding also stops leaf elongation before turgor is affected (Wadman-van Schravendijk and van Andel 1985). A similar change in root signal, rather than changes in leaf turgor, might explain the early reduction in leaf elongation in response to N stress. The present study was undertaken to consider several possible mechanisms by which N stress might reduce growth. In barley and tomato, we examined changes in photosynthesis, water relations, abscisic acid (ABA; a hormone implicated in both N and water stress), and N mobilization from internal stores. Material and methods Seedlings of barley (Hordeum vulgare L. cv. Midas) and tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) were grown in solution culture which supplied all N as NO~- until two leaves had reached full expansion, as described by Chapin et al. (1987). Then, at the start of the experiment (day 0), half of the plants were maintained in complete nutrient solution containing 10 m M NO~-, and half were transferred to a solution of similar osmolarity lacking NO~-. Measurements were conducted at days 0, 2, 5 and 10. All nutrient solutions were replenished at days 0 and 5. At each harvest date, barley plants were subdivided into roots, old leaves (i.e. leaves 1 and 2) and young leaves (the remainder of the shoot). Tomatoes were subdivided into roots and shoots. Dry weights were determined after oven-drying for 48 h at 60 ~ C. On some plants, old and young leaves were subsampled into liquid nitrogen for whole-tissue ABA analysis, as described elsewhere (Chapin et al. 1987). In tomato, old leaves consisted of the leaflets of the two oldest true leaves; young leaves were the leaflets of the two largest expanding leaves. Length of the lamina (barley) or terminal leaflet (tomato) was measured periodically on selected leaves. Root sugar concentrations were measured on whole root axes of barley and tomato plants by boiling in 25 or 50 ml 80% ethanol for 10 rain, then decanting the supernatant. The root was treated with two further volumes of boiling 80% ethanol. The washings were combined with the original extract and, when cool, made up to 50 or 100 ml with 80% methanol. A subsample of 7 ml was evaporated to dryness under vacuum and redissolved in either 2 or 4 ml water. Total sugars were determined by autoanalyzer using a slightly modified version
353
of the sulfuric acid-orcinol method (Shannon 1972); reducing sugars were determined by the copper-bicinchoniate procedure (Mopper and Gindler 1983).
Tissue water content per unit leaf area was calculated from fresh:dry weight subsamples used in A B A analyses (Chapin et al. 1987) and from specific leaf weight (see below). Dried samples were then ground in a Wiley mill. One subsample (0.1 g) was heated in 5 ml distilled water at 90 ~ C for 2 h (mixed thoroughly every 0.5 h), filtered and analyzed for NO~- concentration (Brewer et al. 1966); a second subsample (0.4 g) was digested in selenous-sulfuric acid and analyzed for organic N by micro Kjeldahl using the salicylate-nitroprusside method. Data are expressed on a fresh-weight basis. In vivo nitrate-reductase activity was determined in freshly excised new and old leaves following vacuum infiltration. Samples were incubated in the dark for 1 h at 20 ~ C in 1.5% propan-l-ol containing 50 m M NO~ and 50 m M phosphate, pH 7.0 (Stewart et al. 1973). Leaves were then boiled for 10 min, and N O r concentration was measured on these extracts and on subsamples collected before incubation (Brewer et al. 1966). Leaf water potential was measured by inserting leaves into a pressure chamber. Between times of collection and measurement, leaves were kept in the dark in a plastic bag with wet filter paper. Water absorption rate was measured for intact tomato plants from the decrease in volume of solution culture over 2-h intervals (as compared with volume changes in an aerated solution containing no plant). Photosynthesis and transpiration were measured on intact leaves with a closed-system portable infrared gas analyzer (model 6000; Licor, Lincoln, Neb., USA), fitted with a Vaisala humidity sensor. From these measurements stomatal conductance and leaf internal CO2 concentration were calculated. Each measurement was initiated as soon as the rate of COz depletion in the chamber stabilized, generally within 30 s. Depletion of CO2 during each measurement averaged 20 ppm. All measurements were made at the photon fluence rate (300 lamol.m-Z.s -1, 40(~700 nm), temperature (20 ~ C), and humidity (50%) at which plants were grown in order to estimate photosynthesis under ambient growth conditions. Flow rate was adjusted to maintain constant relative humidity during the measurement period. The area of leaf samples from photosynthetic measurements was measured on a leaf-area meter (Delta T Devices, Burwell, Cambridge, U K ) ; then leaves were dried and weighed to determine specific leaf weight. Fluxes of water (J~), NO~ and total solutes into the xylem under normal growing conditions were estimated for excised root systems by placing them in the solutions in which plants were grown (i.e. roots of N-sufficient plants in complete nutrient solution and roots of N-stressed plants in nutrient solutions of similar osmolarity but lacking N O ~ ) and collecting and analyzing the xylem exudate (Chapin et al. 1981). Osmolarity of the xylem sap and external solution was measured and used to calculate root hydraulic conductance (Lv), as described by Glinka (1980) and Radin and Boyer (1982). The effect of exogenous ABA upon these transport parameters was determined, as described elsewhere (Chapin et al. 1987).
Results
Growth. Relative growth rate (RGR) of N-sufficient controls was higher in tomato (0.21 g . g - a .
354
F.S. Chapin III et al. : Mechanisms of growth response to nitrogen stress
TOMATO
BARLEY 6000
-~ CI 600 0
co
9 5000
o 300 ._A E
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F-
600
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~
0 l DAY 5
::L IJ.J
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4
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~-t
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200 F DAY4
.o-. .o2 4 6 8 AFTER EXCISION
0 L i 2
J .
i
i
J
4
6
8
(h)
Fig. 6. Nitrate flux o f xylem, N O 3 , concentration, and volume o f xylem exudate in excised r o o t s o f barley and t o m a t o exposed to the solutions in which they were grown. D a t a are means 4-SE, n = 4. Statistics as in Fig. 1
T a b l e 2. Effect o f N stress on osmolarity o f xylem sap and hydraulic conductance o f excised roots. D a t a are means _+ SE, n = 4.
Statistics as in Fig. I Species
Barley Tomato
Duration of N stress (d)
5 2 4
Osmolarity (mmol-1 1)
Hydraulic conductance ( p l . g - 1 . h -1 . k P a - 1 )
+N
--N
+N
--N
88.0 _+ 1.7 39.5 _+ 1.9 28.5_+0.5
82.7-t-0.9" 30.8_+1.4" 27,8_+0.3
0.20_+0.01 4.2 _+0.7 8.1 _+1.3
0.13_+0.01 * 1.4 _+0.2* 1.4 _+0.0"
and tomato roots (caused by the small root pool available for transport) and (2) a smaller exudate volume (J~) in tomato immediately following excision. The smaller J~ in tomato was apparently the result of a lower hydraulic conductance (Lp, Table 2). Jv increased during the 6 h after excision in N-stressed roots, partially compensating for the difference in NO 3 flux between N-stressed roots and controls. The differential between initial NO;fluxes in N-stressed and control roots became more pronounced with time (day 4 or 5 versus day 2) and was greater in tomato (Fig. 6) which had the lowest concentration of root NO~ reserves (Fig. 4).
Water-absorption rates of entire plants (groot; Table 1) were 10-fold higher than J~ in excised roots (Fig. 6), indicating that root pressure accounts for only a small proportion of the water transport in the intact plant. Similarly, the rate of organic-N accumulation in the shoot (calculated from Fig. 5) was threefold higher than that observed for the NO~- flux to the xylem of excised roots (Fig. 6), indicating that the transpiration stream enhances NO 3 transport to the shoot. Exogenous ABA generally caused no significant change in NO 3 flux to the xylem, because increases in J~ (in barley N-stressed roots and in tomato N-stressed roots at day 2) tended to dilute
358
F.S. Chapin III et al. : Mechanisms of growth response to nitrogen stress
Table 3. Effect of exogenous ABA on NO 3 flux to xylem, NO 3 concentration, and volume of xylem exudate in excised roots of barley and tomato after varying duration of N stress. " T " indicates 0.1 gM 2-trans-ABA (the biologically inactive isomer); otherwise ABA (the biologically active isomer) was applied at the indicated concentrations (~tM). Data are means _+SE, n =4. Statistics indicate significant differences from 0 ABA addition, as described in Fig. 1 [AB~
0
T
0.01
0.1
1.0
0.53 +0.12 0.14 _+0.04
0.65 +0.15 0.17 +_0.01
0.67 _+0.17 0.16 +0.02
0.50 - 0 . 1 0 0.020 _+0.002
0.73 +_0.13 0.023 +_0.002
0.70 +_0.12 0.028 -+ 0.004
NO 3 flux to xylem ( g m o l . g - 1. h - 1) Barley Day 2 +1 --N
0.41 • 0.04 0.11 _+0.01
Day 5 +N --N
0.68 _+0.08 0.022 -+ 0.004
0.52 _+0.06 0.016_+0.005
Day 2 +N -N
0.84 _+0.08 0.051 _+0.002
0.72 _+0.04 0.051 -+0.003
1.13 +-0.28 0.074 _+0.007 *
Day 4 +N -N
0.65 _+0.08 0.018_+0.001
0.69 -+0.16 0.016 -+ 0.001
0.85 _+0.08 0.028 _+0.005
Tomato
Xylem NO3 concentration (mmol.1 1) Barley Day 2 +N --N
25.4 +_ 1.4 2.6 _+0.3
Day 5 +N --N
21.8 _+ 1.2 0.90+_0.13
21.4 _+1.0 0.65_+0.21
19.2 _+ 1.2 1.07+_0.15
18.4 -+ 1.5 1.11 _+0.02
18.8 -+1.3 1.11-+0.10
13.1 _+0.7 0.19_+0.04
12.8 _+0.5 0.14_+0.003
13.2 +-0.6 0.22_+0.05
25.7 _+0.3 2.5 _+0.5
23.6 -+2.1 2.5 _+0.1
25.7 + 1.3 2.3 +0.1
21.5 • 0.74 _+0.08
25.0 +-2.3 0.65-+0.08
23.1 • 1.2 0.75_+0.11
Tomato Day 2 +N -N Day 4 +N --N
Exudate volume (gl. g - 1.h t) Barley Day 2 +N -N
23_+3 34-+3
Day 5 +N --N
33_+3 24_+ 1
26+_1 25+_0
Day 2 +N --N
136+_17 81_+7
97_+6 77_+5
201 _+27 115_+3"
Day 4 +N --N
87-+9 81_+9
99_+19 91_+4
113+8 98_+10
27_+5 51+_3"
27_+5 72_+4**
21_+5 72_+4**
26_+6 28_+2
33+_8 36+_2*
33_+7 37+ *
Tomato
F.S. Chapin III et al. : Mechanisms of growth response to nitrogen stress
359
BARLEY .
TOMATO .
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Fig. 7. Photosynthesis, stomatal conductance and internal CO2 concentration of barley and tomato leaves of plants grown with and without NO~-. Data are means + S E , n - 1 0 . Statistics as in Fig. 1
exudate N O 3 concentration to give a nearly constant NO~- flux (Table 3). Nitrogen-sufficient roots did not respond to exogenous A B A in either species. The biologically inactive 2-trans-ABA had no significant effect upon any measured parameter.
Gas exchange. Young barley leaves had higher photosynthetic rates than old leaves; in both young and old leaves rates declined with time (Fig. 7). Photosynthetic rate was increased by N stress in young leaves at day 2, reduced by N stress in old leaves by day 5 and reduced by N stress in all leaves by day 10. Stomatal conductance roughly paralleled changes in photosynthetic rate, declining with leaf age, increasing with N stress in young leaves at days 2 and 5, declining with N stress in old leaves at day 5 and declining with N stress in young leaves at day 10. Because of the parallel changes in photosynthesis and stoma-
tal conductance with N stress, leaf internal CO2 concentration (ci) was not affected by N stress (Fig. 7). Specific leaf weight declined only slightly with N stress (Fig. 3), so that patterns of photosynthesis expressed per unit tissue weight were similar to those shown in Fig. 7. Photosynthetic rate was closely correlated with both organic-N content (r = 0.68 **) and stomatal conductance ( r = 0.82**), indicating that the latter was closely adjusted to the changing photosynthetic potential of the leaf. Consequently, ci was not affected by N stress (Fig. 7). Photosynthetic rate in N-sufficient tomatoes was nearly twice that measured in barley, and, unlike barley, showed no consistent change with leaf number or leaf age (Fig. 7). In tomato, photosynthetic rate decreased in response to N stress in old leaves by day 2 and in all leaves by day 5. Stomatal conductance was not closely coupled to photosynthetic rate in tomato, in that it declined with age
360
F.S. C h a p i n III et al. : M e c h a n i s m s o f growth response to nitrogen stress
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