Soil water deficits decrease the internal conductance to CO2 transfer but atmospheric water deficits do not.

The internal conductance to CO2 supply from substomatal cavities to sites of carboxylation poses a large limitation to photosynthesis. It is known that internal conductance is decreased by soil water deficits, but it is not known if it is affected by atmospheric water deficits (i.e. leaf to air vapour pressure deficit, VPD). The aim of this paper was to examine the responses of internal conductance to atmospheric and soil water deficits in seedlings of the evergreen perennial Eucalyptus regnans F. Muell and the herbaceous plants Solanum lycopersicum (formerly Lycopersicon esculentum) Mill. and Phaseolus vulgaris L. Internal conductance was estimated with the variable J method from concurrent measurements of gas exchange and fluorescence. In all three species steady-state stomatal conductance decreased by approximately 30% as VPD increased from 1 kPa to 2 kPa. In no species was internal conductance affected by VPD despite large effects on stomatal conductance. In contrast, soil water deficits decreased stomatal conductance and internal conductance of all three species. Decreases in stomatal and internal conductance under water deficit were proportional, but this proportionality differed among species, and thus the relationship between stomatal and internal conductance differed among species. These findings indicate that soil water deficits affect internal conductance while atmospheric water deficits do not. The reasons for this distinction are unknown but are consistent with soil and atmospheric water deficits having differing effects on leaf physiology and/or root-shoot communication.

Does growth temperature affect the temperature responses of photosynthesis and internal conductance to CO2? A test with Eucalyptus regnans.

Internal conductance to CO(2) transfer from intercellular spaces to chloroplasts (g(i)) poses a major limitation to photosynthesis, but only three studies have investigated the temperature dependance of g(i). The aim of this study was to determine whether acclimation to 15 versus 30 degrees C affects the temperature response of photosynthesis and g(i) in seedlings of the evergreen tree species Eucalyptus regnans F. Muell. Six-month-old seedlings were acclimated to 15 or 30 degrees C for 6 weeks before g(i) was estimated by simultaneous measurements of gas exchange and chlorophyll fluorescence (variable J method). There was little evidence for acclimation of photosynthesis to growth temperature. In seedlings acclimated to either 15 or 30 degrees C, the maximum rate of net photosynthesis peaked at around 30 or 35 degrees C. Such lack of temperature acclimation may be related to the constant day and night temperature acclimation regime, which differed from most other studies in which night temperatures were lower than day temperatures. Internal conductance averaged 0.25 mol m(-2) s(-1) at 25 degrees C and increased threefold from 10 to 35 degrees C. There was some evidence that g(i) was greater in seedlings acclimated to 15 than to 30 degrees C, which resulted in seedlings acclimated to 15 degrees C having, if anything, a smaller relative limitation due to g(i) than seedlings acclimated to 30 degrees C. Stomatal limitations were also smaller in seedlings acclimated to 15 degrees C than in seedlings acclimated to 30 degrees C. Based on chloroplast CO(2) concentration, neither maximum rates of carboxylation nor RuBP-limited rate of electron transport peaked between 10 and 35 degrees C. Both were described well by an Arrhenius function and had similar activation energies (57-70 kJ mol(-1)). These findings confirm previous studies showing g(i) to be positively related to measurement temperature.

Changes in gas exchange versus leaf solutes as a means to cope with summer drought in Eucalyptus marginata.

Two of the ways in which plants cope with water deficits are stomatal closure and "osmotic adjustment". We sought to assess the contributions of these processes to maintenance of leaf hydration in field-grown, 7-year-old Eucalyptus marginata. Plants were exposed to their normal summer drought (controls) or supplied with additional water (irrigated). Irrigation increased photosynthesis by 30% in E. marginata. These increases in photosynthesis were related to an 80% increase in g (s). However, there was no difference in substomatal CO(2) concentrations between treatments, or in chloroplast CO(2) concentrations, as indicated by carbon isotope composition of leaf soluble sugars. This suggests that impaired mesophyll metabolism may partially explain slower rates of photosynthesis in plants exposed to their normal summer drought. There was no difference in concentrations of solutes or osmotic potential between non-irrigated and irrigated individuals, perhaps because relative water content was the same in non-irrigated and irrigated plants due to stomatal sensitivity to water deficits. Irrespective of the absence of osmotic adjustment, analysis of leaf solutes gave a clear indication of the major groups of compounds responsible for maintaining cell osmotic potential. Soluble sugars were three times as abundant as amino acids. Proline, a putatively osmotically active amino acid, contributed less than 1% of total solutes. These patterns of solutes in E. marginata are consistent with a growing body of literature arguing a greater role for carbohydrates and cyclitols and lesser role for amino acids in maintaining osmotic potential. Our data suggest the primary mechanism by which E. marginata coped with drought was partial stomatal closure; however, we cannot discount the possibility of osmotic adjustment under more severe water deficits.

O3 flux-related responsiveness of photosynthesis, respiration, and stomatal conductance of adult Fagus sylvatica to experimentally enhanced free-air O3 exposure.

Knowledge of responses of photosynthesis, respiration, and stomatal conductance to cumulative ozone uptake (COU) is still scarce, and this is particularly the case for adult trees. The effect of ozone (O(3)) exposure on trees was examined with 60-year-old beech trees (FAGUS SYLVATICA) at a forest site of southern Germany. Trees were exposed to the ambient O(3) regime (1 x O(3)) or an experimentally elevated twice-ambient O(3) regime (2 x O(3)). The elevated 2 x O (3) regime was provided by means of a free-air O(3) canopy exposure system. The hypotheses were tested that (1) gas exchange is negatively affected by O(3) and (2) the effects of O(3) are dose-dependent and thus the sizes of differences between treatments are positively related to COU. Gas exchange (light-saturated CO(2) uptake rate A(max), stomatal conductance g (s), maximum rate of carboxylation Vc (max), ribulose-1,5-bisphosphate turnover limited rate of photosynthesis J (max), CO(2) compensation point CP, apparent quantum yield of net CO(2) uptake AQ, carboxylation efficiency CE, day- and nighttime respiration) and chlorophyll fluorescence (electron transfer rate, ETR) were measured IN SITU on attached sun and shade leaves. Measurements were made periodically throughout the growing seasons of 2003 (an exceptionally dry year) and 2004 (a year with average rainfall). In 2004 Vc(max), J(max), and CE were lower in trees receiving 2 x O(3) compared with the ambient O(3) regime (1 x O(3)). Treatment differences in Vc (max), J (max), CE were rather small in 2004 (i.e., parameter levels were lower by 10 - 30 % in 2 x O(3) than 1 x O(3)) and not significant in 2003. In 2004 COU was positively correlated with the difference between treatments in A (max), g (s), and ETR (i.e., consistent with the dose-dependence of O(3)'s deleterious effects). However, in 2003, differences in A(max), g (s), and ETR between the two O(3) regimes were smaller at the end of the dry summer 2003 (i.e., when COU was greatest). The relationship of COU with effects on gas exchange can apparently be complex and, in fact, varied between years and within the growing season. In addition, high doses of O(3) did not always have significant effects on leaf gas exchange. In view of the key findings, both hypotheses were to be rejected.

Uptake of nitrate, ammonium and glycine by plants of Tasmanian wet eucalypt forests.

A central assumption of ecosystem N cycling has been that organic N must be converted to inorganic N to be available for plant uptake, but this has been questioned by recent studies. We examined uptake of nitrate, ammonium and the amino acid glycine in three species from Eucalyptus obliqua L'Her. wet forest in Tasmania, south-eastern Australia, to test the hypothesis that all three species can take up glycine, and to compare rates of glycine uptake with rates of uptake of nitrate and ammonium uptake. The alternative hypothesis that species vary in their preference for nitrate, ammonium and glycine ("niche differentiation") was also examined. Measurements were made on the canopy dominant Eucalyptus obliqua, and two rain forest tree species found in the understory or as sub-dominants of the canopy, Nothofagus cunninghamii (Hook.) Oerst. and Phyllocladus aspleniifolius (Labill.) Hook.f. Nitrogen uptake was examined in situ with attached roots placed in uptake solutions containing equimolar concentrations (100 micromol l(-1)) of (15)N-nitrate, (15)N-ammonium and 2-(13)C(2) (15)N-glycine. Species did not differ in their preference for different forms of N (species x N form interaction, P > 0.05), and thus there was no evidence of niche differentiation. In all species, rates of uptake were highest for ammonium (11 +/- 5 micromol g(DM) (-1) h(-1); mean +/- SD, n = 108), uptake of glycine occurred at less than half this rate (4.4 +/- 2.6 micromol g(DM) (-1) h(-1)), whereas uptake of nitrate occurred at one-tenth of this rate (0.9 +/- 1.2 micromol g(DM) (-1) h(-1)). The strong positive relationship between (15)N and (13)C uptake indicated that at least 72% of glycine-N was taken up intact. These findings indicate the potential for considerable uptake of organic N in the field.

Temperature response of photosynthesis and internal conductance to CO2: results from two independent approaches.

The internal conductance to CO(2) transfer from intercellular spaces to chloroplasts poses a major limitation to photosynthesis, but few studies have investigated its temperature response. The aim of this study was to determine the temperature response of photosynthesis and internal conductance between 10 degrees C and 35 degrees C in seedlings of a deciduous forest tree species, Quercus canariensis. Internal conductance was estimated via simultaneous measurements of gas exchange and chlorophyll fluorescence ("variable J method"). Two of the required parameters, the intercellular photocompensation point (C(i)*) and rate of mitochondrial respiration in the light (R(d)), were estimated by the Laisk method. These were used to calculate the chloroplastic photocompensation point (Gamma*) in a simultaneous equation with g(i). An independent estimate of internal conductance was obtained by a novel curve-fitting method based on the curvature of the initial Rubisco-limited portion of an A/C(i) curve. The temperature responses of the rate of Rubisco carboxylation (V(cmax)) and the RuBP limited rate of electron transport (J(max)) were determined from chloroplastic CO(2) concentrations. The rate of net photosynthesis peaked at 24 degrees C. C(i)* was similar to reports for other species with a C(i)* of 39 micromol mol(-1) at 25 degrees C and an activation energy of 34 kJ mol(-1). Gamma* was very similar to the published temperature response for Spinacia oleracea from 20 degrees C to 35 degrees C, but was slightly greater at 10 degrees C and 15 degrees C. J(max) peaked at 30 degrees C, whereas V(cmax) did not reach a maximum between 10 degrees C and 35 degrees C. Activation energies were 49 kJ mol(-1) for V(cmax) and 100 kJ mol(-1) for J(max). Both methods showed that internal conductance doubled from 10 degrees C to 20 degrees C, and then was nearly temperature-independent from 20 degrees C to 35 degrees C. Hence, the temperature response of internal conductance could not be fitted to an Arrhenius function. The best fit to estimated g(i) was obtained with a three-parameter log normal function (R(2)=0.98), with a maximum g(i) of 0.19 mol m(-2) s(-1) at 29 degrees C.

Potential organic and inorganic N uptake by six Eucalyptus species.

There are no published studies of organic N uptake by species of south-eastern Australia (e.g. Eucalyptus) despite several studies of ecosystem N cycling. This study examines uptake of nitrate, ammonium and glycine (an amino acid) by six species of 16-year-old Eucalyptus growing at two plantations ('common gardens'). By using two plantations, one xeric / oligotrophic and one mesic / eutrophic, I was able to disentangle genotypic from phenotypic differences in preference for N forms. Measurements were made on three separate occasions during spring. N uptake was examined in situ with attached roots placed in uptake solutions containing equimolar 100 µmol L-1 concentrations of 15N-nitrate, 15N-ammonium and 2-13C215N-glycine. Water and KCl extracts were used to determine the relative abundances of nitrate, ammonium and amino acids at the two plantations. Nitrate dominated at the eutrophic site, but was nearly absent at the oligotrophic site. N at the oligotrophic site was dominated by ammonium and amino acids which were present in similar concentrations. The rate of uptake of ammonium (6.3 ± 0.4 µmol g h-1; mean ± s.e., n = 108), was faster than glycine (3.4 ± 0.2), which was faster than nitrate (0.62 ± 0.07). Plant 'preference' for N forms did not vary between sites despite large differences in the relative abundances of N forms (nitrate v. ammonium v. amino acids). Hence, there was little evidence for acclimation of Eucalyptus species to differences in the relative availability of N forms. This study suggests the possibility for considerable organic N uptake in the field. Previous studies of ecosystem N cycling in south-eastern Australia have only examined inorganic N. The N cycle in south-eastern Australia needs to be revisited with a new perspective, one that considers inorganic N and organic N.

Water stress decreases the transfer conductance of Douglas-fir (Pseudotsuga menziesii) seedlings.

We tested the hypothesis that transfer conductance (gi) of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings is reduced by water stress. Seedlings were irrigated with a solution of 25% polyethylene glycol so as to impose water stress rapidly, thereby limiting acclimatory responses. Transfer conductance was measured pre-treatment and post-treatment by two methods. Water stress reduced net photosynthesis by 20-50%. The initial slope of the rate of photosynthesis (A) over the intercellular carbon dioxide (CO2) concentration (Ci) response was reduced by water stress, indicating that reduced photosynthesis was not wholly accounted for by reduced stomatal conductance. The carbon isotope and chlorophyll fluorescence methods both indicated that water stress decreased gi. From isotopic measurements with 1% O2, gi was 0.076 +/- 0.009 (mean +/- SE) mol m(-2) s(-1) in well-watered seedlings and 0.044 +/- 0.004 mol m(-2) s(-1) in water-stressed seedlings. Fluorescence estimates of gi were 0.08 +/- 0.01 mol m(-2) s(-1) in well-watered seedlings and 0.044 +/- 0.004 mol m(-2) s(-1) in water-stressed seedlings. The drought-induced reduction in gi was responsible for the reduction in slope of the A/Ci response, and thus there was no difference in the slope of the A over the chloroplastic CO2 concentration (Cc) response between treatments and no indication of impaired mesophyll metabolism. These data illustrate that impairments of mesophyll metabolism can be revealed only from analysis of the A/Cc response.

Photosynthetic responses and N allocation in Douglas-fir needles following a brief pulse of nutrients.

The temporal distribution of soil nutrients is heterogeneous, and thus the uptake, storage and later remobilization of brief nutrient pulses may be critical for growth in nutrient-limited habitats. We investigated the response of photosynthesis and the major nitrogen (N) fractions in needles of 2-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings to a 15-day nutrient pulse (containing 250 ppm N). The nutrient pulse (N pulse) was imposed in late July, toward the end of the seedlings' third growing season, and subsequent changes in photosynthesis and needle N fractions were examined over the following 3 months. Needles are sites of photosynthesis and putative storage organs. Thus we tested two hypotheses: (1) N from the N pulse is quickly synthesized from soluble non-protein N into soluble proteins, especially Rubisco, and (2) the N pulse increases photosynthetic rates and thus growth. We also examined an alternative hypothesis that Rubisco functions also as a storage protein, in which case we would predict increases in amount of Rubisco in response to the N pulse without concomitant increases in photosynthesis. Soluble non-protein N was the most dynamic N pool and may have constituted a temporary storage reservoir; however, the quantitative significance of soluble non-protein N is questionable because this pool was at most only 7% of total N. Concentrations of Rubisco were unaffected by the N-pulse treatment and there was little evidence that Rubisco served as a storage protein. Nutrient-pulse seedlings added twice as much dry mass as controls during the 3 months post-treatment (Warren et al. 2003a). Over the same period, the maximum rate of light-saturated photosynthesis (A(max)) declined to low rates in control seedlings, whereas A(max) increased in N-pulse seedlings. Nevertheless, treatment and temporal trends in N and Rubisco content per unit area were poorly related to A(max), and it seems likely that photosynthesis was limited by additional factors, perhaps thylakoid proteins or an inadequate supply of other nutrients.

Response of Douglas-fir seedlings to a brief pulse of 15N-labeled nutrients.

The temporal distribution of soil nutrients is heterogeneous, and thus the uptake, storage and later remobilization of brief nutrient pulses may be critical for growth in nutrient-limited habitats. We investigated the response of 2-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings receiving a low nutrient supply to a 15-day nutrient pulse (containing 250 ppm nitrogen (N) as 10 atom % 15NH4 15NO3). The nutrient pulse was imposed in late July, toward the end of the seedlings' third growing season, and subsequent changes in dry mass and N content over the following 3 months were determined from destructive harvests. We tested three hypotheses: (1) N from the nutrient pulse is rapidly assimilated and accumulated primarily in needles and roots; (2) this accumulated N is later remobilized to support new growth; and (3) the nutrient pulse leads to a larger second flush of shoot growth. Seedlings increased their N content by 175 mg (67%) in response to the nutrient pulse. Nitrogen was taken up preferentially into younger tissues, especially the secondary flush and current-year roots. Immediately after the nutrient pulse, tissue N concentrations were high and supported subsequent increases in dry mass. Over 3 months, seedlings receiving the nutrient pulse added twice as much dry mass as control seedlings, and even after 3 months of growth, N concentrations remained greater than in controls. Current-year and older needles were the only components whose dry mass did not increase over this period. The nutrient pulse increased the size of the second flush, but it was still a minor component of increments in dry mass (approximately 10% of the total dry mass increment) and N content (23%). The relatively modest increases in N content during autumn could be accounted for by soil uptake and there was no evidence that N was remobilized to support growth of new tissues. Short-term (15 days) elevated N uptake led to sustained growth in the long term (> 3 months), and thus growth rate was to a large extent decoupled from current nutrient supply.

Responses of gas exchange to reversible changes in whole-plant transpiration rate in two conifer species.

This study examined the autonomy of branches with respect to the control of transpiration (E) in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and western red cedar (Thuja plicata Donn) seedlings. Experiments were conducted on whole seedlings in a gas exchange system with a dual-cuvette that permitted independent manipulation and measurement of E in the upper and lower cuvettes. The value of E in one cuvette was manipulated by varying vapor pressure deficit (D) between 2.2 and 0.2 kPa, whereas D in the other cuvette was held at 2.2 kPa. Reducing D, while increasing stomatal conductance (gs), resulted in an overall decrease in E. In western red cedar, this decrease was almost threefold, and in Douglas-fir, approximately fourfold. In well-watered western red cedar, a reduction of whole-plant E by 46% (brought about by reducing D in the upper cuvette) resulted in a 12% increase in gs, a 12% increase in E and a 7% increase in net assimilation (A) of untreated foliage in the lower cuvette. Responses of gs, E and A of untreated foliage were similar irrespective of whether foliage was at the top or bottom of the seedling. When D in the treatment cuvette was restored to 2.2 kPa, gs, E and A of foliage in the untreated cuvette returned to pretreatment values. In contrast, in well-watered Douglas-fir, there was almost no change in gs, E or A of untreated foliage in one cuvette when D in the other cuvette was reduced, causing a 52% reduction in whole-plant E. However, similar manipulations on drought-stressed Douglas-fir led to 7-19% increases in gs, E and A of untreated foliage. In well-watered western red cedar, daytime leaf water potential (Psil) was maintained near -0.9 MPa over a wide range of D, whereas Psil of Douglas-fir decreased from -1.2 to -1.5 MPa as D increased. The tighter (isohydric) regulation of Psil in western red cedar may partly explain its greater stomatal response to D and variation in whole-plant E compared with Douglas-fir. In response to a reduction in E, measured increases in Psil and gs of unmanipulated foliage were less than predicted by a model assuming complete hydraulic connectivity of foliage. Our results suggest the foliage of both species is partially autonomous with respect to water.

Capillary electrophoresis for the determination of major amino acids and sugars in foliage: application to the nitrogen nutrition of Sclerophyllous species.

Amino acids and sugars are probably the most commonly measured solutes in plant fluids and tissue extracts. Chromatographic techniques used for the measurement of such solutes require complex derivatization procedures, analysis times are long and separate analyses are required for sugars and amino acids. Two methods were developed for the analysis of underivatized sugars and amino acids by capillary electrophoresis (CE). Separation of a range of sugars and amino acids was achieved in under 30 min, with good reproducibility and linearity. In general, there was close agreement between amino acid analyses by CE and HPLC with post-column derivatization. An alternative, more rapid method was optimized for the common neutral sugars. Separation of a mixture of fructose, glucose, sucrose, and fucose (internal standard) was achieved in less than 5 min. How the source of N applied (nitrate or ammonium) and its concentration (8.0 or 0.5 mM) affects the amino acid and sugar composition of leaves from Banksia grandis Willd. and Hakea prostrata R. Br. was investigated. The amino acid pool of Banksia and Hakea were dominated by seven amino acids (aspartic acid, glutamic acid, asparagine, glutamine, serine, proline, and arginine). Of these, asparagaine and glutamine dominated at low N-supply, whereas at high N-supply the concentration of arginine increased and dominated amino-N. Plants grown with nitrate had a greater concentration of proline relative to plants with ammonium. In Banksia the concentration of amides was greatest and arginine least with a nitrate N-source, whereas in Hakea amides were least and arginine greatest with nitrate N-source. The concentration of sugars was greater in Banksia than Hakea and in both species at greater N-supply.

Trade-offs between the persistence of foliage and productivity in two Pinus species.

We investigated interspecific variation in leaf lifespan (persistence) and consequent differences in leaf biochemistry, anatomy, morphology, patterns of whole-tree carbon allocation and stand productivity. We tested the hypothesis that a species with short-lived foliage, Pinus radiata D. Don (mean leaf lifespan 2.5 years), grows faster than P. pinaster Ait., a species with more persistent foliage (leaf lifespan 5.6 years), and that the faster growth rate of P. radiata is associated with a greater allocation of nitrogen and carbon to photosynthetic tissues across a range of scales. In fully sunlit foliage, the proportion of leaf N in the major photosynthetic enzyme Rubisco (ribulose-1, 5-bisphosphate carboxylase) was greater in P. radiata than in P. pinaster, and, in mid-canopy foliage, the proportion of leaf N in thylakoid proteins was greater in P. radiata. A lesser proportion of needle cross-sectional area was occupied by structural tissue in P. radiata compared to P. pinaster. Foliage mass in stands of P. radiata was 9.7 t ha-1 compared with 18.2 t ha-1 in P. pinaster while leaf area index of both species was similar at 4.6 m2 m-2, owing to the compensating effect of differences in specific leaf area. Hence trade-offs between persistence and productivity were apparent as interspecific differences in patterns of whole-tree carbon allocation, needle morphology, anatomy and biochemistry. However, these interspecific differences did not translate into differences at the stand scale since rates of biomass accumulation were similar in both species (P. radiata 6.9±0.9 kg year-1 tree-1; P. pinaster 7.4±0.9 kg year-1 tree-1). The similarities in performance at larger scales suggest that leaf area index (and radiation interception) determines growth and productivity.