• The relationship between night-time respiration rate and carbohydrate supply are measured in two alpine perennials, Bistorta bistortoides and Campanula rotundifolia.
• Natural populations of B. bistortoides and C. rotundifolia were subjected to high and low light treatments. During the following night, respiration rate and carbohydrate content of leaves were measured.
• At the beginning of the dark period, leaf carbohydrate concentrations were significantly lower in plants exposed to lower irradiance on the previous day. The night-time respiration rate in leaves of both species was rarely affected by the previous days’ irradiance; over the course of the night, respiration rate remained unchanged in C. rotundifolia and decreased, but not consistently, in B. bistortoides. No significant interaction was found between irradiance and the night-time pattern of carbohydrate concentration or respiration rate. Nocturnal carbohydrate export was positively correlated with the previous days’ irradiance in leaves of C. rotundifolia only.
• Leaf respiration rate is uncoupled from carbohydrate supply in leaves of B. bistortoides and C. rotundifolia, possibly allowing more carbohydrate to be exported to underground storage organs.
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The effect of irradiance on carbohydrate concentration and night-time respiration rate is well documented in crop species grown in the controlled conditions of the laboratory. But, does daytime irradiance affect night-time respiration rate in natural populations of plants? Results of the few studies conducted provide conflicting evidence (Noguchi et al., 1996; Noguchi & Terashima, 1997; Atkin et al., 2000). Studies in controlled environments are different from field studies in at least two ways. The irradiance provided in a controlled environment is generally fixed to one or two irradiances per day and temperature is either held constant between light and dark periods or altered by 7°C at most . Plants growing in natural populations may be subject to extremely variable photon flux densities (PFD) which change on a scale of minutes to days. In addition, diurnal temperature fluctuation is rarely constant in the field and may vary by 20°C on the alpine tundra where this study was conducted. Temperature change affects respiration rate and metabolism and export of carbohydrates (Farrar, 1988) such that, at low temperatures, less carbohydrate is metabolized by respiration (Farrar, 1980; Azcon-Bieto & Osmond, 1983) and sucrose and starch accumulate in leaves and other tissues (Farrar, 1980, 1988).
The purpose of this study was to measure night-time respiration rate in the field in order to test the following hypotheses. (1) Respiration rate decreases over the night-time period as assimilate concentration decreases. The decrease in assimilate is the result of both respiratory loss and assimilate export to sink tissues. (2) Night-time respiration and assimilate export are dependent on the photon flux density during the previous light period. In addition, night-time carbohydrate export rate was estimated. Respiration rate and carbohydrate concentration were measured during the night in two species of plants, Bistorta bistortoides and Campanula rotundifolia, on the alpine tundra. Alpine species have different carbon allocation strategies than most crop plants. Approx. 90% of the biomass of an alpine plant is below-ground, creating a strong below-ground sink for carbohydrates.
Materials and Methods
Two herbaceous perennials, Bistorta bistortoides (Pursh) Small (Polygonaceae) and Campanula rotundifolia L. (Campanulaceae), were studied at the Long-term Ecological Research site on Niwot Ridge in the alpine ecosystem of the Colorado Rockies. In B. bistortoides leaves, respiration rates and carbohydrate concentrations were measured on three nights in the summer of 1997 and on one night in the summer of 1998. In addition, respiration rates and carbohydrate concentrations were measured in leaves of the fructan-accumulator C. rotundifolia over the course of two nights in 1998.
Plants were subjected to one of several light treatments on the day previous to the night-time measurements. Some plants were exposed to full sunlight. Subpopulations of plants (40–60 individuals) were exposed to reduced irradiance. Irradiance was reduced by placing 1 m square pieces of shadecloth 35 cm above the soil surface. In 1997, two shadecloth treatments were applied to subpopulations of B. bistortoides. The darker treatment consisted of two layers of shadecloth and will be refered to as ‘low light’ while the lighter treatment (one layer of shade cloth) will be refered to as ‘intermediate light’. Irradiance was measured in full sunlight and under the shadecloth treatments at 20–30 min intervals from approx. 09:00 hours until sunset with a quantum sensor attached to a data logger (Li-Cor Inc., Lincoln, NE, USA, model 1000). In 1997, ambient temperature in full sunlight and in each of the shadecloth treatments was recorded every 60–90 min with a single temperature logger (Onset Computer Corporation, Pocasset, MA, USA, model HOBO) in each treatment placed 10 cm off of the ground.
In 1997, two densities of shadecloth were used to lower irradiance. Average irradiance was calculated from 09:00 hours until sunset. The intermediate light treatment reduced the irradiance to 30–40% of full sunlight and the low light treatment by 85–90% (Table 1). Because no significant difference was found between the two shadecloth treatments in terms of carbohydrate concentration or respiration rate, only the less dense shadecloth treatment (referred to as intermediate light) was used in 1998. Light environment varied considerably between days (Table 1). Air temperature was reduced by 1–8°C in the low light treatment and by 0–4°C in the intermediate light treatment and the reduction in temperature varied between collection days (Table 1). Reductions in temperature caused by the treatments were generally not correlated with carbohydrate concentration at sunset.
Table 1. Irradiance and temperature in full sunlight and under the shadecloth treatments for each collection day
Leaves were collected from plants from each treatment at sunset, between 23:00 hours and 24:00 hours, and before sunrise. The leaves were immediately divided into portions for respiration measurements and carbohydrate analysis. Tissues used for the respiration measurements were stored at 5°C until the respiration measurements were made. Respiration rate was measured within 2 h of leaf collection. The tissue used for carbohydrate analysis was immediately placed on dry ice and kept frozen until the carbohydrate extractions were made.
Leaf respiration rate was measured in air with oxygen electrodes (Hansatech Corporation, UK) following the methods of Delieu & Walker (1981). The temperature of the electrodes was maintained at 5 or 15°C with circulating water baths (Brinkman Model RM6, Fisher Scientific, Pittsburgh, PA, USA, model 9105). Respiration rates were measured at 5 and 15°C. After the respiration rate was measured, the leaves were stored on dry ice until they could be dried in a drying oven in the laboratory. The leaves were placed in a 100°C drying oven for 4 h and then stored in a 55°C drying oven until the dry weight was recorded. Respiration rates were calculated as nmol O2 consumed per second per gram of dry tissue weight (nmol O2 s−1 g−1 d. wt).
The extraction procedures and assays used to measure carbohydrate pools in the leaves differed between the two species because the storage form of carbohydrate varied between the species. B. bistortoides accumulates starch while C. rotundifolia accumulates fructan, a polymer of fructose. Leaf tissue used for carbohydrate measurements was kept in a −70°C freezer until the carbohydrates were extracted.
The first portion of the extraction was similar between the two species. Soluble carbohydrates, including short polymer length fructans, were extracted twice with of 85% (v/v) ethanol. Tissues were sonicated (Fisher Scientific model FS15) and centrifuged (Eppendorf Scientific, Westburg, NY, USA, model 5402). The ethanol extract and pellet were dried in a heating block (Fisher Scientific) at 35°C. Soluble carbohydrates were isolated from lipids and pigments in the acqueous phase of a chloroform : water extraction. The pellet from each sample was retained for extraction of starch or long-chain fructans (approx. 20 fructose units or longer).
Deionized water was added to both the B. bistortoides and C. rotundifolia pellets. The B. bistortoides pellets were autoclaved to solublize starch. The C. rotundifolia pellets were boiled for 10 min to solublize the > 20 fructose unit polymer fructans (refered to as long polymer (LP) fructans).
All carbohydrate assays were made in 96 well microtiter plates and read on a microtiter spectrophotometer (Molecular Devices, Sunnyvale, CA, USA, model Vmax). Glucose, fructose, and sucrose were measured with a modification of the methods of (Cairns, 1987). In B. bistortoides leaves, glucose, fructose and sucrose were measured with a glucose assay kit purchased from Sigma, The Wood-lands, TX, USA, (Part 115-A). This is a colourimetric assay which utilizes iodonitrotetrazolium chloride (INT) and phenazine methosulphate (PMS) to detect glucose concentrations. The reaction mixture also contains hexokinase which phosphorylates both glucose and fructose. The assay is performed in a stepwise manner within a single well of the microtiter plate. In B. bistortoides leaves, glucose was measured first with fresh dye reagent. Fructose was then converted to glucose with the addition of phosphoglucose isomerase (PGI) (Boehringer-Mannheim, Indianapolis, IN, USA, 127 396, 700 U ml−1 in 0.2 M Hepes, pH 7.8). Lastly, sucrose was converted to glucose and fructose with the addition of invertase (Sigma I-4504, 8250 U ml−1 in 0.1 M citrate, pH = 6.0). In C. rotundifolia leaves, glucose and fructose were assayed in the same manner as B. bistortoides leaves, but invertase was replaced with sucrase (Megazyme, Co. Wicklow, UK, Fructan Assay Kit, 5 U ml−1 in 0.1 M sodium malate, pH 6.5) because invertase catalyses the removal of fructose units from fructans. After the addition of each enzyme, the plate was incubated for 20 min at 37°C and then read spectrophotometrically at 492 nm.
Both short polymer (SP) length (< 20 fructose units) and long polymer length (> 20 fructose units) fructans were converted to fructose and glucose with fructanase (Megazyme, Fructan Assay Kit, 2000 U ml−1 in 0.1 M sodium acetate, pH 4.5) and sucrase. Fructose was converted to glucose with phosphoglucose isomerase. A glucose assay kit (Sigma A-115) was used to measure the concentration of fructan (in glucose equivalents) as already described.
Starch from the B. bistortoides leaves was converted to glucose with amyloglucosidase (Boehringer-Mannheim, 2000 U ml−1 in 0.05 M sodium acetate, pH 4.8). Glucose equivalents were measured with MBTH (Sigma), DMAB (Sigma), and PGO enzymes (Sigma) incubated with the sample. Absorbance was read at 595 nm to obtain the glucose equivalents of starch.
All carbohydrate concentrations were calculated on a dry weight basis by converting fresh weight to dry weight with a fresh weight : dry weight ratio.
The maximum possible carbohydrate export during the night was estimated by subtracting the amount of carbohydrate lost through respiratory flux from the change in substrate concentration between sunset and sunrise. The loss of carbohydrate from respiratory flux was estimated by first log transforming the respiration rates measured at 5 and 15°C. A linear equation was then fitted to these data with temperature as the independent variable and the log of respiration rate as the dependent variable. In this way, respiration rate could be estimated at the ambient night-time temperature at the site. The temperature sensitivity of respiration (Q10) generally increases with decreasing temperature and such a change would result in a overestimation of respiratory loss at low temperatures. The bias in our estimates is likely to be small because ambient night-time temperature was often between 5 and 15°C and only dropped a few degrees below 5°C. Respiration rate was converted from nanomoles of oxygen to nanomoles of carbon dioxide by assuming that the respiratory quotient is constant at 1.0 (Salisbury & Ross, 1992). The loss of carbon by respiratory processes between sunset and sunrise could then be estimated by multiplying the night-time rate by the amount of time elapsed from sunset to sunrise. The change in carbohydrate concentration was calculated by subtracting the total carbohydrate at sunrise from the total carbohydrate at sunset. In these calculations, we assumed that carbohydrates were not converted to other forms of organic molecules. If carbohydrates were transformed into other organic molecules (ex. proteins or lipids) then a decrease in carbohydrates would occur through processes other than respiration and export. Some carbohydrate is likely used for the maintenance of proteins and lipids. The input, though, is probably small in the later part of the growing season when the measurements were made because the leaves are no longer growing. The loss of carbon by respiration was then subtracted from the change in carbohydrate content of the leaf to estimate the maximum possible export rate.
One-way ANOVAs were utilized to compare the change in sugar concentration, starch or fructan concentration, and respiration rate over-night. Two-way ANOVAs were used to compare the effect of light on night-time carbohydrate concentration and respiration rate and the possible interaction of daytime irradiance with night-time patterns of carboydrate concentration and respiration rate. No significant interaction was found between irradiance and night-time pattern of carbohydrate concentration or respiration rate. All statistics were performed in Microsoft Excel, Redmond, WA, USA or Jumpin (SAS Institute, Cary, NC, USA).
The shadecloth treatments significantly reduced the night-time concentrations of glucose, fructose, and sucrose (hereafter called soluble sugars) in B. bistortoides leaves on 2 days (Fig. 1, Table 2). Soluble sugar concentration in B. bistortoides leaves decreased during the night on 2 nights in leaves exposed to full sunlight but rarely changed in leaves from the shadecloth treatments (Fig. 1, Table 2). The shadecloth treatments significantly reduced the night-time concentration of starch in the B. bistortoides leaves on all collection nights (Fig. 2, Table 2). Starch concentrations rarely decreased significantly over the night in B. bistortoides leaves.
Table 2. P-values from one-way ANOVAs for night-time Bistorta bistortoides leaf glucose, fructose, sucrose and starch concentrations comparing differences in concentration among light treatments and changes in concentration through the night
July 31, 1997
Aug 14, 1997
Aug 3, 1998
ns signifies that the P-value was > 0.05.
**Total sugars. na, refers to glucose plus fructose plus sucrose concentration.
In general, night-time B. bistortoides leaf respiration rate was not significantly lowered by reduced irradiance on the previous day (Fig. 3, Table 3). In addition, B. bistortoides leaf respiration rate rarely decreased significantly during the night when measured at 5°C and did not decrease when measured at 15°C (Table 3). In addition, the Q10 of respiration rate was not altered by time or treatment (data not shown).
Table 3. P-values from one-way ANOVAs comparing the effect of the previous day’s irradiance on night-time Bistorta bistortoides leaf respiration (treatment effect) and the change in Bistorta bistortoides leaf respiration through the night (night-time effect)
Night-time soluble sugar concentration of C. rotundifolia leaves did not differ significantly between treatments (Fig. 4,Table 4). C. rotundifolia leaf soluble sugar concentration only decreased significantly through the night in leaves exposed to full sunlight on August 26. The shadecloth treatment significantly reduced the night-time concentration of fructans in the C. rotundifolia leaves on only one collection night (Fig. 5, Table 4). Leaf fructan concentration rarely decreased significantly during the night (Fig. 5, Table 4).
Table 4. P-values from one-way ANOVAs for Campanula rotundifolia leaf glucose, fructose, sucrose, and fructan content comparing differences in concentration among light treatments and changes in concentration through the night
Aug 3, 1998
Aug 26, 1998
ns signifies that the P-value was > 0.05.
**Total sugars refers to glucose plus fructose plus sucrose concentration.
Respiration rate in the C. rotundifolia leaves did not vary with the previous day’s irradiance at either measurement temperature (Fig. 6, Table 5). Respiration rate did not decrease over the night-time period when measured at 5°C (Fig. 6a,c). When measured at 15°C, respiration rate decreased significantly through the night in leaves from both treatments on August 3, 1998 (Table 5). Q10 did not change significantly with time or treatment on either night (data not shown). This would seem to contradict the finding that respiration rate decreased at night at 15°C but not at 5°C on August 3, 1998. At 5°C, respiration rate tends to have a higher variance than at 15°C and changes in respiration rate at night may not be significant, in part, because of the higher variance. At 5°C on August 3, 1998, respiration rate does decrease during the night but the decrease is not significant.
Table 5. P-values from ANOVAs comparing the effect of the previous day’s irradiance on night-time Campanula rotundifolia leaf respiration (treatment effect) and the change in Campanula rotundifolia leaf respiration through the night (night-time effect)
Night-time rates of carbohydrate export were estimated by subtracting modelled rates of total night-time respiration from the total change in carbohydrate concentration over-night. Total night-time respiratory carbon loss was estimated by calculating respiration rates at ambient temperatures (Fig. 7) using experimentally determined temperature sensitivity curves. Calculated night-time carbohydrate export rates were not correlated with light treatment in B. bistortoides leaves (Table 6). On two nights (July 30, 1997 and August 3, 1998), carbohydrate export was higher in leaves which received full sunlight on the previous day than in leaves which were exposed to reduced irradiance. Night-time carbohydrate export was 2–3 times higher from C. rotundifolia leaves exposed to full sunlight than from leaves subjected to the shadecloth treatment (Table 6).
Table 6. Night-time export rates (µmol carbon100 mg−1 of leaf d. wt) from leaves exposed to different irradiance on the previous day
July 30, 1997
Aug 13, 1997
Aug 3, 1998
Export rates were calculated from 19:00 hours until 05:00 hours on the following morning.
**not applicable, measurements not made at low light on this date.
The previous day’s irradiance occasionally had an effect on night-time respiration rate in B. bistortoides leaves and did not affect night-time respiration rate in C. rotundifolia leaves. These results are in contrast to the findings of numerous other studies conducted on crop plants (Hansen & Jensen, 1977; Azcon-Bieto & Osmond, 1983; Reddy et al., 1991; Noguchi & Terashima, 1997). These results are also unexpected in B. bistortoides leaves because leaf carbohydrate content (both soluble sugars and starch) was significantly and positively correlated with irradiance. Thus, in B. bistortoides leaves, an increase in irradiance which resulted in an increase in carbohydrate content did not lead to an increase in night-time leaf respiration rate as seen in past studies (Breeze & Elston, 1978; Azcon-Bieto & Osmond, 1983; Mullen & Koller, 1988b; Noguchi et al., 1996; Noguchi & Terashima, 1997). The effect of irradiance on C. rotundifolia leaf carbohydrate concentration was small and it was not surprising that night-time respiration rate was unaffected by irradiance.
B. bistortoides leaf respiration rate rarely decreased significantly during the night and C. rotundifolia leaf respiration rate decreased significantly on only one collection date. Soluble sugar content occasionally decreased at night and starch content always decreased at night in the B. bistortoides leaves exposed to full sunlight. Soluble sugar and fructan content of C. rotundifolia leaves also often decreased at night (although not significantly in most cases). Thus, respiration rate and carbohydrate concentration do not seem to be coupled in the leaves of these two species.
A lack of correlation between these two variables may be the result of regulation of respiration in leaves of these species by a factor other than carbohydrate supply. Respiration rate is controlled by a number of variables, including the supply of substrates such as carbohydrate and diphosphate adenosine (ADP), the activity of enzymes in the respiratory pathway which are controlled by enzyme concentration and allosteric regulation, and by the energy demand of the tissue. In a series of studies by Noguchi et al. (1996) and Noguchi & Terashima (1997) on spinach, a sun species, and Alocasia odora, a shade species, the effects of irradiance, exogenously supplied sucrose, and FCCP (an uncoupler of mitochondrial oxidative phophorylation) on respiration rate were measured during a period of darkness. In the crop species, spinach, night-time respiration rate was proportional to daytime irradiance and respiration rate decreased at night. When exogenous sucrose was added to the spinach leaves, respiration rate could be maintained throughout the dark period at levels similar to those found immediately postillumination. The addition of FCCP, though, had no effect on respiration rate in spinach leaves at any time during the night. In contrast, night-time rates of respiration in leaves of Alocasia were not altered by daytime irradiance, time of night, or exogenous sucrose. The addition of FCCP did result in an increase in respiration rate at all times of the night. The authors concluded that respiration rate in spinach is limited by substrate availability while in Alocasia is limited by demand for ATP (i.e. energy demand is low). Low energy demand would result in the inhibition of glycolysis and the tricarboxylic acid (TCA) cycle and in ADP limitation of oxidative phosphorylation (Wiskich, 1980; Lambers et al., 1998). Azcon-Bieto et al. (1983a) found that respiration rate in wheat leaves was controlled by different factors immediately after illumination and 14 h postillumination. Immediately following illumination, respiration rate could be stimulated by sucrose and glycine only in the presence of FCCP while after 14 h of darkness respiration was stimulated by sucrose and glycine but only slightly by FCCP. Respiration rate had also decreased during the dark period. The authors concluded that, in wheat leaves, respiration rate immediately following a long period of photosynthesis was limited by oxidative phophorylation but after hours of darkness adenylate supply was less important than substrate supply for glycolysis and the TCA cycle. These studies clearly indicate that respiration rate is affected by a number of biochemical factors which vary in a complex manner with species, substrate limitation, and enzyme regulation.
In the alpine plants studied, the insensitivity of respiration to carbohydrate concentration could be the result of low night-time temperatures and differences in the temperature sensitivity of carbohydrate metabolism, respiration, and carbohydrate export. During the summer, night-time temperatures on the alpine tundra generally range between −2 and +8°C while daytime temperatures are between +8 and 25°C (C.L. McCutchan, unpublished). A number of studies have shown that night-time respiration rate and/or carbohydrate export are reduced at low temperatures (Breeze & Elston, 1978; Farrar, 1980; Azcon-Bieto & Osmond, 1983). In wheat leaves, postillumination rates of respiration remained high for longer in plants exposed to cold temperatures (Azcon-Bieto & Osmond, 1983). Additionally, a sharp drop in temperature at night may decrease some biochemical processes more than others. Sucrose and fructan synthesis seem to be less temperature sensitive than respiration and carbohydrate export (Farrar, 1988). Sucrose phosphate synthase and the fructan synthesizing enzymes, sucrose-sucrose fructosyl transferase and fructan-fructan fructosyl transferase, have Q10s (2–10°C) between 1.4 and 1.7 (Pollock, 1986a,b). The Q10 of respiration in many species is often approx. 2 (10–20°C) and is likely higher at lower temperatures (Larigauderie & Körner, 1995). The relative temperature sensitivities of these two processes would result in higher rates of sucrose and fructan production relative to respiration at low temperatures which would result in more carbohydrate available for export. Little is known about the temperature sensitivity of carbohydrate export from leaves relative to respiration and carbohydrate metabolism within the leaves. If export is less sensitive to changes in temperature than respiration, export would be favoured over respiration at low temperatures.
The uncoupling of leaf respiration from carbohydrate concentration may be advantageous in alpine perennial plants. Much of the biomass of an alpine plant is found in underground storage tissues (roots and rhizomes) which are essential to the survival, growth, and reproduction of alpine perennials. Organic compounds necessary for the rapid flush of growth at the beginning of the growing season are stored in these underground tissues. This storage allows leaves to be produced quickly and seeds to be set before the end of the growing season (Billings & Mooney, 1968). Wyka (1999) found that shading and defoliation treatments in the alpine species Oxytropis sericea resulted in a decrease in storage carbohydrates, infructescenses, and leaf biomass in the following year. Storage organs may also provide insurance against years with poor summer growing conditions. Storage organs are likely to be very strong sinks for carbohydrates and, for this reason, may result in high carbohydrate export rates from leaves. This would be especially true during the late summer and early autumn when our observations were made. During this period, storage pools are replenished in preparation for the following spring (Mooney & Billings, 1960; Hadley & Bliss, 1964; Wyka, 1999). By decoupling leaf respiration rate from carbohydrate concentration, more carbohydrate would be available for export to storage organs.
If we assume that a decrease in leaf carbohydrate concentration during the night is the result of only respiratory loss and export (i.e. transformation to other organic compounds does not occur), then < 40% of the carbon lost at night is consumed by respiratory processes and > 60% is exported. The proportion of carbohydrate exported is slightly lower than that reported for Solanum tuberosum (potato) and Phaseolus vulgaris (bean) (67–85%) (Bouma et al., 1995). In addition, Bouma et al. (1995) calculated that much of the respiratory flux seen at night is associated with export. B. bistortoides leaves did not show a correlation between daytime irradiance and night-time export rate as has been seen in Glycine max (soybean) leaves (Fader & Koller, 1983; Mullen & Koller, 1988a). In contrast, carbohydrate export rate from C. rotundifolia leaves was positively correlated with daytime irradiance.
This study has shown that night-time respiratory rates in natural populations of plants may not have the same response to irradiance or the same relationship to carbohydrate supply as those reported previously for crop species. Most of the species used in these past studies are annual plants with limited allocation to storage pools. The results of our study make clear the need to examine the functional relationship between respiration rate and carbohydrate supply in plants with a variety of carbon allocation patterns and varying demands on the photosynthate pool.
Logistical support was provided by the NSF supported Niwot Ridge Long-Term Ecological Research project and the University of Colorado Mountain Research Station. The authors wish to thank William Adams for the use of oxygen electrode equipment, Barbara Demmig-Adams, Steve Hand, Jeff Mitton, and two anonymous reviewers for reviewing a draft of the manuscript, and Amy Keller and Lorené Martin for assistance in the field.