Journal of Experimental Botany, Vol. 52, No. 354, pp. 123-131,
January 2001
© 2001 Oxford University Press
Original Papers |
Gas exchange by pods and subtending leaves and internal recycling of CO2 by pods of chickpea (Cicer arietinum L.) subjected to water deficits
1 Centre for Legumes in Mediterranean Agriculture, University of Western Australia, Nedlands, WA 6907, Australia
2 CSIRO Plant Industry, Private Bag No. 5, Wembley, WA 6913, Australia
3 Institute of Natural Resources, Massey University, Palmerston North, New Zealand
Received 25 April 2000; Accepted 17 August 2000
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Abstract |
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Terminal drought markedly reduces leaf photosynthesis of chickpea (Cicer arietinum L.) during seed filling. A study was initiated to determine whether photosynthesis and internal recycling of CO2 by the pods can compensate for the low rate of photosynthesis in leaves under water deficits. The influence of water deficits on the rates of photosynthesis and transpiration of pods and subtending leaves in chickpea (cv. Sona) was investigated in two naturally-lit, temperature-controlled glasshouses. At values of photosynthetically active radiation (PAR) of 900 µmol m-2 s-1 and higher, the rate of net photosynthesis of subtending leaves of 10-d-old pods was 24 and 6 µmol m-2 s-1 in the well-watered (WW) and water-stressed (WS) plants when the covered-leaf water potential (
) was -0.6 and -1.4 MPa, respectively. Leaf photosynthesis further decreased to 4.5 and 0.5 µmol m-2 s-1 as decreased to -2.3 and -3.3 MPa, respectively. At 900–1500 µmol m-2 s-1 PAR, the net photosynthetic rate of 10-d-old pods was 0.9–1.0 µmol m-2 s-1 in the WW plants and was -0.1 to -0.8 µmol m-2 s-1 in the WS plants. The photosynthetic rates of both pods and subtending leaves decreased with age, but the rate of transpiration of the pods increased with age. The rates of respiration and net photosynthesis inside the pods were estimated by measuring the changes in the internal concentration of CO2 of covered and uncovered pods during the day. Both the WW and WS pods had similar values of internal net photosynthesis, but the WS pods showed significantly higher rates of respiration suggesting that the WS pods had higher gross photosynthetic rates than the WW pods, particularly in the late afternoon. When 13CO2 was injected into the gas space inside the pod, nearly 80% of the labelled carbon 24 h after injection was observed in the pod wall in both the WW and WS plants. After 144 h the proportion of 13C in the seed had increased from 19% to 32% in both treatments. The results suggest that internal recycling of CO2 inside the pod may assist in maintaining seed filling in water-stressed chickpea. Key words: Pod photosynthesis, leaf photosynthesis, CO2 recycling, respiration, 13C labelling of pods.
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Introduction |
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The rates of leaf photosynthesis in chickpea are markedly reduced by water deficits. Field measurements have shown that they are below 5 µmol m-2 s-1 throughout seed filling in a Mediterranean-type climate (Leport et al., 1998
, 1999). However, seed filling continues under drought conditions, albeit for a shorter duration (Davies et al., 1999). Recent studies have suggested that the redistribution of dry matter from stems and leaves of chickpea could provide up to 60% of the final seed weight (Leport et al., 1999), and that the pod wall could contribute 9–15% of the final seed weight in chickpea grown under terminal drought (Davies et al., 1999). More detailed studies using 13C showed that less than 20% of the seed carbon in chickpea came from pre-podding stored assimilates when plants were water-stressed, proportions significantly less than those calculated from changes in dry weight (Davies et al., 2000). Other studies using 14CO2 suggested that the leaves were the primary source of carbon for seed growth, particularly the leaf subtending the pod (Singh and Pandey, 1980). If the rate of photosynthesis of this subtending leaf is very low, and this needs to be verified, and redistribution of carbon from other plant parts is also low, the question arises as to the source of carbon to maintain seed filling in water-stressed chickpea.
Previous studies have shown that during the early stages of seed development pod walls of chickpea can fix CO2 under well-irradiated conditions, but generate large losses of CO2 through respiration in the dark (Sheoran et al., 1987). In field pea (Pisum sativum), it has been shown that pod photosynthesis fixed only a small amount of atmospheric CO2 between 6 d and 30 d after flowering, but under well-watered conditions re-fixed most of the 1500 to 15 000 µl l-1of CO2 respired during the day by the seeds inside the pod cavity (Flinn et al., 1977). Similar results were also reported in soybean (Glycine max) (Sambo et al., 1977) and white lupin (Lupinus albus) (Atkins and Flinn, 1978), indicating that re-fixing respired CO2 inside the pod for use by the growing seed may be an important mechanism for reducing losses of carbon.
Although photosynthesis by the external pod wall has been suggested as contributing to seed growth in chickpea (Sheoran et al., 1987), Leport et al. were unable to measure any gas exchange by pods in the field (Leport et al., 1999). Moreover, the effects of water deficits on the gas exchange by pods and CO2 recycling inside the pod remain unanswered. The aim of the present study was to determine the influence of water deficits on the net CO2 exchange of both pods and subtending leaves at different ages using more sensitive equipment than that of other authors (Leport et al., 1999). Additionally, the role of the pod wall and seed coat in internal CO2 fixation and their potential to reduce carbon losses during seed filling was evaluated in well-watered and water-stressed chickpea.
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Materials and methods |
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Four experiments were conducted. In Experiment 1, the gas exchange of pods and subtending leaves at a range of light intensities was measured with the increase in water deficits and age to establish whether the response of pods was similar to that of leaves. In Experiment 2, diurnal changes in net CO2 exchange of pods and internal levels of CO2 within the pods were measured. Experiment 3 aimed at measuring the diurnal changes in photosynthesis and respiration within the pod, and Experiment 4 examined the fixation and distribution of labelled CO2 between the pod wall and seed over the first 6 d after labelling.
Plant material, soil and fertilizer
The four experiments were conducted in two naturally-lit glasshouses located at Floreat Park, Perth, Western Australia (31°57' S, 115°51' E) at various times of year in 1999. The day/night temperatures were maintained at 22/11 °C in one glasshouse used for Experiments 1, 3 and 4, and at 22/15 °C in the other glasshouse used for Experiment 2. Chickpeas (Cicer arietinum L. cv. Sona) were grown in free-draining polyvinylchloride pots (diameter 15 cm and depth 40 cm). The seeds were sown in a commercial potting mix for germination, and 1 week later one seedling was transplanted to each pot. At transplanting, the roots were inoculated with commercial group N Bradyrhizobium.
Experiments 1, 2 and 4 used the same soil and fertilizer. The soil was a reddish brown, sandy clay loam (Calcic Haploxeralf, pH 7.0 in 0.01 M CaCl2) collected from an area of undisturbed native soil near Merredin, Western Australia. The soil was sieved to remove any large aggregates and pieces of organic matter. The top 10 cm of soil in the pots was a mixture of Merredin soil and yellow sand at a ratio of 1:1 to prevent the soil surface crusting, compared to a ratio of 9:1 of the same mixture for the lower profile. Both the upper and lower soils were mixed with fertilizers (g kg-1): 0.15 KNO3, 0.14 NH4NO3, 0.2 Ca(NO3)2, 0.15 triple superphosphate and 0.6 Micromax (g kg-1: 1.2 Fe, 0.25 Mn, 0.1 Zn, 0.05 Cu, 0.01 B, 0.005 Mo, and 1.5 S). In Experiment 3, the soil was the surface 10 cm of a loamy sand (Xeric Psamment, pH 4.6 in 0.01 M CaCl2) with 92% sand and 4% clay collected from a field site near Wongan Hills, Western Australia. The soil was mixed before transplanting with fertilizers (g kg-1): 0.2 superphosphate (amended with Cu, Zn and Mo) and 0.5 muriate of potash.
All pods were tagged when visible (3 mm) in order to select pods of the same age at sampling. Plants began to set pods about 8 weeks after sowing. At pod set, the subtending leaf was about 15-d-old and flowering occurred 10 d prior to pod set. Stomatal densities of leaves, and internal and external pod walls were counted under a microscope (x400) from acrylic surface impressions from five different pods or leaves. The surfaces were sprayed with a thin coating of acrylic film (artists fixative) and then transferred to a microscope slide on transparent tape (Clemens and Jones, 1978).
Watering treatments
The soil in all four experiments was watered to field capacity at transplanting and then watered to saturation twice a week until the two watering regimes were applied 70 d after sowing (DAS) in Experiments 1, 2 and 4 and 75 DAS in Experiment 3. Water was withheld from half of the plants, hereafter called water-stressed (WS), while the rest was kept watered as previously, hereafter called well-watered (WW). In Experiments 1, 2 and 4, the soil volume and soil type, and the use of only one plant per pot, ensured a slow and steady development of water deficits. In these experiments pod and leaf measurements were made between 11 d and 28 d after withholding water. In Experiment 3 water deficits developed more rapidly in the sandy soil and measurements were made 7 d after withholding water.
Measurements
Experiment 1 – Light response of net photosynthesis and transpiration:
The rates of net photosynthesis and transpiration of pods and subtending leaves were measured using a CIRAS-1 portable photosynthesis system (PPS, Hitchin, UK) with a standard leaf chamber which was large enough to enclose a single pod. The system had a light unit which provided levels of photosynthetically active radiation (PAR) from 10 to 1500 µmol m-2 s-1. Net photosynthetic measurements were made at PARs of 10, 100, 300, 600, 900, 1200, and 1500 µmol m-2 s-1. Both the pod and leaf photosynthetic rates were calculated on a projected area basis. The leaf area:leaf oven-dry weight ratio was measured on a representative set of leaves in order to calculate the rate of photosynthesis on a leaf dry weight basis. Pods of three different ages (10, 20 and 30 d after pod set) were selected from both the WW and WS plants with each measurement being replicated three times on different plants. The rates of net photosynthesis and transpiration of the pods and subtending leaves were measured 13, 18 and 26 d after withholding water. On the same days the water potential () of fully expanded leaves that had been covered with aluminium foil on the late afternoon of the previous day was measured near dawn using the pressure chamber technique, following the precautions described previously (Turner, 1988). Before measurement the proximal four leaflets were removed and the leaf midrib was inserted into the pressure chamber. Covered-leaf is considered to be equivalent to the in the stem at the point of attachment to the leaf (Begg and Turner, 1970) and has been shown to be similar to the of the pod wall in chickpea (Shackel and Turner, 2000).
Experiment 2 – Pod net photosynthesis and internal CO2 concentration:
Twenty-one days after withholding water, the rates of net photosynthesis of 10–12-d-old pods were measured at 06.00 h (predawn), 09.00 h, 12.00 h, 15.00 h, and 18.00 h (sunset) on a clear sunny day using the CIRAS-1 portable photosynthesis system. At the same time, the CO2 concentration inside the pods of similar age was measured using a CI-301 (CID Inc., Moscow, ID, USA) CO2 analyser (Atkins and Pate, 1977). The pods in the spaced chickpea plants were fully exposed to light for most of the day. Before measurement, the analyser was calibrated with four concentrations of CO2 ranging from 0 to 1040 µl l-1. In 10–12-d-old pods, the internal gas space is about 400 µl. During sampling, the needle of a 1 ml syringe was inserted into the gas space in the proximal end of a pod, and a 200 µl sample of gas was collected and immediately injected into the analyser. Predawn covered leaf was measured using the pressure chamber technique as in Experiment 1. All the measurements were replicated five times on different plants.
Experiment 3 – Net photosynthesis and respiration inside the pod:
The internal CO2 concentration of six 10-d-old pods per treatment was measured from predawn (05.30 h) to sunset (18.30 h) at intervals of approximately 2 h on a clear sunny day, using a new set of pods each time and with the same methods as described in Experiment 2. The decrease in CO2 concentration inside the pods between any two successive measurements was used to estimate the rate of net photosynthesis of the internal pod wall and seed coat for that period. At the same time, a different group of six pods was covered with aluminium foil for 2 h and the internal CO2 concentration measured in order to estimate the rate of respiration by the internal pod wall and seed coat for that period. In order to establish the influence of CO2 concentration on the rates of net and gross photosynthesis inside the pod, another group of six pods that had been covered for 2 h (resulting in high internal CO2 concentrations) were uncovered and the CO2 concentration inside the pod sampled. Other pods were then exposed to sunlight for 2 h before sampling. After the measurement of CO2 concentration inside the pod, each pod was harvested and its weight was measured after being dried in a forced-draught oven at 70 °C for 72 h. The rates of respiration and net photosynthesis inside the pod were calculated on a dry weight basis and the sum of the two rates was used to calculate the rate of gross photosynthesis. It was assumed that the rate of respiration in the light was the same as that in the dark. The of covered leaves was measured at predawn, 09.00 h, 12.00 h, 15.00 h, and sunset by the pressure chamber technique as in Experiment 1 with each measurement being replicated five times.
Experiment 4 – 13CO2 feeding inside the pod:
On day 11 after withholding water the covered leaf water potential was measured prior to dawn as in Experiment 1. At 09.00–10.00 h, 0.5 ml of 99.9% atom 13C-labelled CO2 was fed into the cavity of 10–12-d-old pods of WW and WS plants. At feeding, a hole was punctured in the basal end of a pod followed by injection of 13CO2 in the proximal end using a 1 ml syringe, and both holes were immediately sealed with a small drop of glue that had no visible short-term or long-term effects on the pod wall. The pods and subtending leaves were harvested at 24, 72 and 144 h after 13CO2 feeding and then quickly freeze-dried. After the pods had been separated into pod wall and seed, the samples were weighed and ground to less than 1 mm for analysis of 13C and C by mass spectrometry (VG Micromass Sira 10) after Dumas combustion (Europa C/N analyser).
Statistical analyses
The pots were randomly arranged on benches in the glasshouse and were moved weekly to ensure that positional effects were removed. The rates of net photosynthesis and transpiration of pods and their subtending leaves were analysed by a four-way ANOVA using the water treatment (WW, WS), organ (pod, leaf), age (10, 20, 30 d) and light (10–1500 µmol m-2 s-1) as main effects. The rates of respiration and net photosynthesis inside the pod were analysed using a one-way ANOVA with the water treatments as the main effect. GENSTAT 5.0 Release 3.2 was used for all ANOVA's.
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Results |
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Pod and leaf gas exchange
Fully-expanded chickpea leaves had a stomatal density of 126±6 mm-2 compared to 31±3 mm-2 in the external pod wall and no stomata in the internal pod wall.
Thirteen days after withholding water in Experiment 1, the covered-leaf near dawn was -1.4 MPa in the WS plants and -0.6 MPa in the WW plants. The rates of net photosynthesis of 10-d-old pods were 0.9–1.0 µmol m-2 s-1 at PARs of 900–1500 µmol m-2 s-1 in the WW plants, and -0.1 to -0.3 µmol m-2 s-1 in the WS plants (Fig. 1a). In contrast, the net photosynthetic rate of the subtending leaves, which were 25-d-old at the time of measurement, increased with increasing PAR and reached 24 and 6 µmol m-2 s-1 at values of PAR above 900 µmol m-2 s-1 in the WW and WS plants, respectively (Fig. 1b). As the fell to -2.3 and -3.3 MPa with time in the WS plants, the pods respired and their subtending leaves fixed CO2 at the rates of 4.5 and 0.5 µmol m-2 s-1, respectively.
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In the WW plants the rate of pod transpiration was 0.55 mmol m-2 s-1 at PARs of 1200 to 1500 µmol m-2 s-1 and decreased to half of that when the covered-leaf
fell to -1.4 MPa (Fig. 1c
). In contrast, the rate of leaf transpiration in the WW chickpeas reached a maximum value of 3.4 mmol m-2 s-1 at a PAR of 900 µmol m-2 s-1, but decreased to 1.0 mmol m-2 s-1 when the dropped to -1.4 MPa (Fig. 1d). Both pods and subtending leaves had lower transpiration rates with the further development of water deficits.
The rates of net photosynthesis and transpiration of the pods and subtending leaves were also affected by age. While 10- and 20-d-old pods had positive rates of photosynthesis at high values of PAR, 30-d-old pods always respired irrespective of PAR (Fig. 2a). Subtending leaves of 10- and 20-d-old pods, which were 25-d-old and 35-d-old at the time of measurement, had similar light response curves for CO2 fixation, but the 45-d-old leaves showed light saturation at a PAR of 600 µmol m-2 s-1 and the maximum rate of net photosynthesis was reduced to half that in the younger leaves (Fig. 2b). The rate of leaf transpiration was also significantly lower (one-third) in the old leaves compared to that in the young leaves (Fig. 2d). In contrast, the rate of pod transpiration was the highest in the 30-d-old pods and lowest in the 10-d-old pods (Fig. 2c).
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Diurnal changes in pod net photosynthesis and internal CO2 levels
Twenty-one days after withholding water in Experiment 2, predawn leaf was -0.7 MPa in the WW plants and -1.7 MPa in the WS plants. At 06.00 h (predawn) 10–12-d-old pods were respiring, but from 09.00–15.00 h the WW pods fixed CO2 at rates between 0.3 and 1.0 µmol m-2 s-1 (Fig. 3a), within the range of the standard errors in Experiment 1. In the WS plants, the rate of net photosynthesis of the pods was lower than that of the WW pods near midday. At 18.00 h (sunset), the pods were again respiring at rates greater than those at predawn, consistent with results in field pea (Atkins and Pate, 1977) and presumably because the glasshouse was warmer at 18.00 h (day temperature) than at 06.00 h (night temperature). The CO2 concentration inside the pods (Fig. 3b) was almost the inverse of the net photosynthesis of the pod measured by external gas exchange. At predawn the CO2 concentration inside the WW and WS pods was about 4500 µl l-1, but decreased to near zero at 09.00 h. The internal CO2 concentration then increased and was higher in the WS than the WW pods. At sunset, the CO2 concentration inside the WS pods was 11 000 µl l-1, but only 7000 µl l-1 in the WW pods. Similar results were obtained in a further experiment.
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) chickpea. The measurements were taken from 10–12-d-old pods. Bars give ±SE of the mean (n=5).
Diurnal changes in respiration and photosynthesis inside the pod
Seven days after withholding water in Experiment 3, the predawn leaf was -0.6 and -1.8 MPa in covered leaves and decreased to -0.8 and -2.3 MPa during the day in the WW and WS plants, respectively (Fig. 4a). The internal rate of respiration of 10-d-old pods was higher in the WS plants than the pods of the WW plants throughout the day, particularly in the afternoon (Fig. 4b). The rate of net photosynthesis inside the pods was 0.10–0.14 µmol g-1 s-1 from predawn to 08.00 h with the WS plants being higher than the WW plants (Fig. 4c). During the rest of the day, the rate of net photosynthesis inside the pods was either negative or low (0.01–0.02 µmol g-1 s-1) and was not significantly different between the WW and WS pods (Fig. 4c). As a consequence of the higher rates of respiration in the WS pods, the pods had higher calculated rates of gross photosynthesis inside the pods than the WW pods throughout the day (Fig. 4d). To determine the effect of high CO2 concentrations on the internal pod photosynthesis, the concentration of CO2 inside the pod was elevated by covering the pods for 2 h prior to the measurements of photosynthesis. The pods with elevated initial levels of CO2 had higher net and gross photosynthetic rates inside the pods in both the WW and WS plants (data not shown). The data from both the WW and WS pods with normal and elevated initial levels of CO2 showed that there was a positive linear relationship (r2=0.81) between the estimated rate of gross photosynthesis and the initial CO2 concentration inside the pods (Fig. 5). The rate of gross photosynthesis was always higher at the same internal CO2 concentration in the WS than WW pods, possibly due to the greater seed size (0.263±0.003 g seed-1 in the WS and 0.174± 0.001 g seed-1 in the WW chickpeas) and faster rate of growth in the WS seeds than the WW seeds.
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, 
) prior to the measurement and were sampled from both well-watered (•,
Allocation of 13CO2 inside the pod
Eleven days after withholding water, the predawn covered- leaf was -0.5 MPa for the WW plants and -1.4 MPa for the WS plants. Twenty-four hours after 0.5 ml of 13CO2 was fed into the cavity of 10–12-d-old chickpea pods in Experiment 4, the excess 13C was similar at 55.6±4.8 and 58.3±6.1 µg in the WW and WS pods, respectively, but no measurable 13C was observed in the subtending leaves. At this time the proportion of 13C in the pod wall was 78% and 80% in the WW and WS pods, while the proportion in the seed was 22% and 20% in the WW and WS pods, respectively (Fig. 6). Two days later a greater proportion of the 13C had moved to the seeds, and this trend continued so that by 6 d after feeding only 65% and 64% of the 13C was in the pod wall, 31% and 34% in the seed, and 4% and 2% in the leaves in the WW and WS pods, respectively. There were no significant differences between the treatments in 13C fixation or redistribution inside the pods.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The present study showed that leaf net CO2 exchange decreased with both a reduction in water potential and leaf age and that the leaves were less responsive to light when water-stressed. These results are consistent with those observed previously, showing that when the midday leaf
in rainfed chickpea fell below -3 MPa leaf photosynthesis decreased to a tenth of its maximum (Leport et al., 1998, 1999). The decrease in leaf photosynthesis with water stress appeared to be largely mediated through stomatal closure as shown from the transpiration data. In contrast to a previous study with less sensitive gas exchange equipment which showed no net CO2 exchange by pods in the field (Leport et al., 1999), this study found that the net CO2 exchange of young, well-irradiated and well-watered pods was positive at values of PAR above 200 µmol m-2 s-1. However, the rate of pod respiration increased with age and the response of the pods to light decreased with water stress so that older pods, especially when water stressed, had negative rates of net CO2 exchange. Stomata were observed on the external wall of the pods, although these were fewer in number than those on the leaves. The transpiration data showed that the stomata on the pod wall were responsive to light and water deficits in a similar manner to those on the leaves. However, in contrast to leaf stomata, the stomata on the external pod wall became more leaky with age leading to higher rates of transpiration and poorer water use efficiencies in old compared to young pods.
While the young and well-watered pods fixed CO2 from the atmosphere outside the pod, the rates of gas exchange were at best only 5% of those in subtending leaves. This suggests that the gas exchange by the pod is only a minor contributor to seed filling in chickpea. Saxena and Sheldrake also came to the same conclusion from experiments in which chickpea pods were exposed to full sunlight during seed filling (Saxena and Sheldrake, 1980). According to Flinn et al., the fruit's respiratory output at night in the well-watered field pea was always in excess of its daytime gain of CO2 from the atmosphere so that overall it was in negative balance for carbon throughout its life (Flinn et al., 1977). Low carbon fixation by pods has also been reported in common bean (Crookston et al., 1974), and rapeseed (Singh, 1995). Sheoran et al. found that covering the pod with aluminium foil from day 3 after flowering reduced seed dry weight by 20% and concluded that the pod CO2 exchange contributed 20% to the seed yield (Sheoran et al., 1987). This conclusion assumes that all the CO2 uptake by the pod is utilized for seed growth, whereas in early pod development most of the dry matter is used for the growth of pod wall (Davies et al., 1999). In addition, when the pod is covered, not only fixation of atmospheric CO2 is inhibited, but also any CO2 recycling inside the pod would be reduced.
The measurements in this study indicate that the gas exchange with the atmosphere by the pods is unlikely to compensate for the stress-induced reduction in current assimilate for seed growth. As a previous study has shown that the carbon accumulated during vegetative growth by the pod wall, leaves, stems, and roots and redistributed to the seed contributed only 20% of the carbon in the seed (Davies et al., 2000), there is still a considerable proportion of carbon needed for seed growth that is unaccounted for from these sources. Previous studies have suggested that the inner epidermis of the pod wall in well-watered pulses and the embryo of rapeseed is effective in refixing CO2 respired by the seeds (Flinn and Pate, 1970; Flinn et al., 1977; Sheoran et al., 1987; King et al., 1998), thereby reducing losses of carbon by respiration. In common bean, the total CO2-fixing capacity through recycling within the pod was estimated to be 26% of that of the subtending leaf (Crookston et al., 1974), and in field pea the inner pod wall was estimated to be capable of refixing 66% of the CO2 released by the seeds into the gas space in the light (Atkins et al., 1977). However, studies have not previously been undertaken in pods of water-stressed pulses. In Experiment 2, the CO2 concentration inside the pods changed diurnally, showing high values at 06.00 h (predawn) and 18.00 h (sunset), but lower values during the day particularly in the morning. The decrease of CO2 concentration inside the pods during the day clearly resulted from CO2 fixation within the pod and not by the leakage to the external atmosphere since the pod showed positive net CO2 gain from the atmosphere at times when the CO2 concentration inside the pod was decreasing. The decrease in CO2 between 06.00 h and 09.00 h in Experiment 2 indicates that the pods fixed 0.9 µg carbon per pod during this 3 h period. The fixation at this rate occurred in both the WW and WS plants. Indeed, the rate of CO2 recycling (gross photosynthesis) inside the pod of 0.7–1.0 µmol g-1 s-1 near midday in the WW and WS plants, respectively, compares favourably with the rate of net photosynthesis of 0.8 µmol g-1 s-1 in the leaves of the WW chickpea when measured at the same time of day and expressed on the same dry weight basis.
The similarity in carbon fixation inside the pod of WW and WS plants was also seen after 13CO2 was injected into the cavity of young pods, suggesting that the recycling of CO2 in the pod was unaffected when chickpea plants were subjected to water-stress. Indeed, the detailed diurnal studies of CO2 concentrations inside the pod in Experiment 3 showed that gross photosynthesis inside the pods was at least as high in the WS than as the WW plants, possibly as a result of the higher internal rates of respiration arising from the faster rates of initial seed growth in the WS pods (Davies et al., 1999). Covering the pods to generate high initial CO2 concentrations inside the pods resulted in high CO2 fixation rates in both the WW and WS plants. The linear relationship between gross photosynthesis and initial CO2 concentration inside the pods (Fig. 5) suggests that the internal CO2 recycling was very efficient and the low rate of respiration of the external pod wall in the dark (Fig. 1) suggests that there is a high resistance to CO2 diffusion within the pod wall itself. The recycling of CO2 inside the pod under water-stressed conditions presumably reduced carbon losses to the atmosphere and increased the utilization of carbon, allowing continued seed growth even though the plants were subjected to terminal drought. However, the re-fixation of CO2 respired by the seeds depends on a supply of carbon from elsewhere, such as the redistribution from the leaves and stems (Davies et al., 2000) and from the low but positive rates of photosynthesis in the leaves of water-stressed chickpea that were observed in this study and in the field (Leport et al., 1998, 1999).
The efficiency of CO2 recycling within the pods, even when plants were water-stressed, may be related to the morphology and water relations of the pod and seed. The high density of chloroplasts in the inner epidermis, the thin cuticle and the dome-shaped outer contours of the epidermal cells of the internal pod wall in field pea have been interpreted as specializations for photoassimilation of CO2 from the pod cavity (Atkins et al., 1977). While the inner epidermis of the chickpea pod wall does not have any stomata on its surface and the data suggest that there is a high resistance to diffusion of CO2 in the pod wall, the 13C study showed that the pod wall initially fixed about 80% of the carbon and over time this carbon was redistributed to the seed. The redistribution of 10% of the 13C from the pod wall to the seed over the first 6 d supports the previous observation that pod walls contribute to seed filling during rapid seed growth in chickpea, particularly in water-stressed chickpea (Davies et al., 1999).
In light of the fact that the pod wall has water potentials similar to those in the leaves of WS plants and much lower than those in the seed (Shackel and Turner, 2000), the observation that nearly 80% of the carbon is fixed by the pod wall in WS as well as WW chickpeas suggests that low water potentials do not inhibit carbon fixation in the inner pod wall. Alternatively, considerable variation may exist in water potentials across the pod wall in the water-stressed chickpea that are not detected by the pressure chamber technique and not observed previously (Shackel and Turner, 2000). These authors only measured the turgor of the cells about 400 µm from the outer epidermis of the pod. The apoplastic space would need to be near full saturation for the seed coat turgor to be maintained (Shackel and Turner, 1998). Therefore, the cells in the pod wall adjacent to the apoplastic space where fixation likely took place (Atkins et al., 1977) may have been considerably higher in water potential than the average of the pod wall measured by the pressure chamber technique or that measured by the pressure probe near the outer epidermis. The variation in water status across the pod wall and the site of fixation of CO2 inside the pod wall are worthy of further investigation.
This study has shown that while water stress suppressed the net CO2 exchange with the atmosphere by both leaves and pods during seed filling, high rates of CO2 recycling inside the pods may help to compensate for the low rates of photosynthesis in the leaves and provide an important source of carbon for seed growth, additional to the previously-reported carbon redistribution from pod walls, leaves and stems, in water-stressed chickpea (Leport et al., 1999; Davies et al., 1999, 2000).
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Acknowledgments |
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We are grateful to Dr Don White for the use of the CIRAS-1 portable photosynthesis system. We also thank Jens Berger for statistical assistance, Christiane Ludwig, Elaine Smith, Mike Barr, and Rebecca Kenney for their technical assistance and Drs KHM Siddique, RJ French, RT Furbank, Z Rengel, and SL Davies for comments on the manuscript. MHB thanks CSIRO for the award of a McMaster Fellowship and the Massey University for the provision of leave.
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Notes |
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4 To whom correspondence should be addressed at CSIRO. Fax: +61 8 9387 8991. E-mail: n.turner{at}ccmar.csiro.au
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References |
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Atkins CA, Flinn AM.1978. Carbon dioxide fixation in the carbon economy of developing seeds of Lupinus albus (L.). Plant Physiology62, 486–490.
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