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Carbon distribution within the plant and rhizosphere for Lolium perenne subjected to anaerobic soil conditions
5011 Bwl. Bwchrm. Vol. 22. No. 5. pp. 643-647. hnted m Great Britam. All nghu reserved
1990
W38-0717.90 S3.00 + 0.00 Copyright C 1990 Pcrgamon Press plc
CARBON DISTRIBUTION WITHIN THE PLANT AND RHIZOSPHERE FOR LOLIUM PERENNE SUBJECTED TO ANAEROBIC SOIL CONDITIONS A. A. MEHARG* Department
of Soil Science. The University
and K. KlLLHAMt
of Aberdeen.
(Accep~prrJ I5 Junuury
Meston
Walk,
Aberdeen
AB9 ZUE.
Scotland
1990)
Summary-Perennial ryegrass was subjected to a range of anaerobic treatments. The distribution of C within the plant was determined by pulse labclling the shoots with “C
INTRODUCTION
0: tension around plant roots causes many complex chanpcs in plant mctnbolic proccsscs and damages root cell structure. Anaerobic mctaholism of photoassimilatcs is much less ctficicnt in terms of cncrgy mol *’ substrate than aerobic mctaholism (Smuckcr. 1984). leading to C starvation within the plant roots (Vartapctian et nl., 1978). When plant roots arc placed in an anaerobic cnvironmcnt the trunslocation of C from shoots to roots is inhibited (Nuritdinov and Vartapctian, 1976). which cxaccrbates C starvation. Anaerobic metabolism in plant roots products (through glycolysis) products such as ethanol. acctaldchydc and lactate. Thcsc compounds arc toxic to plant roots and arc thought to relax ccl1 mcmbrancs, thus increasing the loss of C from plant roots into the rhizosphcrc. Thcsc compounds released may provide substrates for pdthogcnic micro-organisms, which further damage the plant (Crawford, 1978; Smuckcr and Erickson. 1976. 1987). Studies to invcstigatc C distribution in plants where the roots have been subjected to anaerobic stress have been carried out in solution culture [e.g. Schumacher and Smuckcr (1985); Minchin and McNaughton (1984); Wicdcnroth and Poskuta (1981); Nuritdinov and Vartapctian (1976)] or in aseptic mist chambers [e.g. Smuckcr and Erickson (1976. 1987)j. Relating solution culture studies of the rhizosphcrc to soil-grown plants has usually littlc or no rclcvancc (Bowen, 1980). Our purpose was to study the photoassimilation and distribution of a “C-CO: pulse label fixed by pcrcnnial rycgrass grown Rcduccd
‘Present address: Department of Biological Sciences. University of Exeter. Hatherly Laboratories. Prince of Wales Road. Exeter EX4 4P.S. England. tTo whom all correspondence should be addressed.
in soil, whcrc the roots of the plant were subjcctcd to a range of anaerobic trcatmcnts. MATERIAIS
ANI) hlETIIOI)S
Soil
The soil used in the cxpcrimcnt was a sandy loam (Tarvcs scrics) Tillycorthic, N.E. Scotland, sampled to a depth of 2Ocm. Some characteristics of the soil were: 3.7% organic C [dctcrmincd by wet digestion, Dalal (1979)j; 256 mg biomass C kg-’ o.d. soil using the fumigation-incubation method of Jcnkinson and Powlson (1976) and pH 6.3 (using a 2.5: I H,O-soil slurry). Plunt
luhdling
and monitoring
chunkhrrs
A plant growth chamber (Fig. I) was designed in which grass plants could bc labellcd using “C-CO1. with subsequent monitoring of root-soil respiration. The base (root-soil chamber) was a section of PVC tubing (length, 21 cm; o.d., 8.3 cm; i.d., 7.3 cm) which was capped at one end and had three ports on one side at 5cm spacings. Glass tubes were inserted through holes bored in No. 20 silicone suba seals in the top and bottom ports. The central port was fitted with a No. 20 silicone suba seal. A PVC disc with a hole in the centrc, through which a section of a 5 ml syringe barrel was placed, was sealed into position just below the top of the base of the monitoring chamber. using high vacuum silicone grease. The disc was scaled into position before pulse labclling of the plants. The shoot chamber was a length of clear perspcx tubing (length, 28 cm; o.d., 8.3 cm; i.d.. 7 cm), capped at one end. fitted with plastic nozzles, which served as air inlets or outlets. The top section was lathed at its base to fit tightly onto the base section (Fig. I) and scaled with high vacuum silicone grease.
A. A. MEHARO and K. KILLHAM growth chambers were checked to ensure that there were no leaks either externally, or between the root-soil and shoot chambers. This was done by pumping air into the root-soil chamber at IOOml min-’ and air outlets of both root-soil and shoot chambers monitored to ensure no air was escaping into the shoot chamber. Plant culture
Fig. I. Design of plant-soil growth chambers. (I) Soil-root chamber; (2) shoot chamber; (2) partition between shoot and soil-root chamber: (4) air from pump; (5) suba-scaled watcrinp port; (6) air to alkaline trap: (7) air from pump; (4) air to alkaline trap: (9) plant joint: (IO) lathed port.
The “C-CO2 was introduccd to the shoot chamber using a suba scaled. quick-tit flask conncctcd by a side-arm to the shoot chamber. Labcllcd CO2 was gcncratcd by injecting “C-labcllcd Na,CO, into concontrated lactic acid. After sufhcicnt time (2 h) for plants to assimilate tho label. ambient air was pumped continuously through the shoot chamber, and the “C-CO? being removed by bubbling exhaust gases through 5 ml I M NaOH in a IO ml test tube. The NaOH traps were changed daily. To monitor root-soil respiration, shoot and root-soil chambers wcrc isolated using a PVC disc with thr grass planted through the syringe barrel. The shoots and root-soil were separated from each other using high vacuum silicone grease around the base of the grass shoots and between the shoots and the sides of the syringe barrel. Moist air was then pumped continuously into the plant growth chamber through both shoot and root-soil chambers at a flow rate of 24 ml min-‘. and respired “C-CO, trapped in 5 ml I M NaOH in a IO ml test tube. Air pumped through the shoot chamber was passed through the alkaline trap to prevent the release of the label into the atmosphere. Shoot respiration data wcrc not reported as shoot rcspircd and non-assimilated label could not be partitioned in the early stage of the cxpcrimcnts. Moisture content of the soil used in this cxpcrimcnt was initially a ficld capacity. At harvest, soil water potential was -2.8 kPa. Thcsc water potentials would be expcricnccd by plants growing under field conditions. Although continually pumping of the root-soil chamber with air will have contributed to the loss of soil moisture the soil water potential data suggested the plants wcrc not subjected to advcrsc soil conditions (0 to -2.8 kPa). Air inlet tubes were fitted with sterilized mcmbranc tiltcr units. The plant
Lo&m perenne (Preference) was grown for 5 days in sand irrigated with a quarter-strength Hoaglands nutrient solution. following germination (4 days) in glass Petri dishes. lined vvith damp filter paper. The ryegrass plants were grown under a light bank of 8 x 85 watt fluorescent tubes (light intensity 20,000 lux) with a I2 h photoperiod, with a maximum day-time temperature of 25°C and a minimum nighttime temperature of ZO’C. Randomly selected plants (26 mg air dry weight) were then transferred to 53 pm nylon mesh tubes containing loosely-packed moist soil (moisture content 35.7%. soil water potential -0.49 kPa). and planted at a density of 5 plants per tube. Nylon mesh tubes were constructed from 6 by 20 cm rectangles of the mesh folded in half and the long edge and one of the short cdgcs heat scaled using a hot soldering iron. The tubes wcrc then placed in the root-soil chamber and soil (950 p. of the same moisture content as that used in the tubes) was packed around the tube to hold it in a central position and the PVC disc placed in position on the top of the soil. The plants were then grown in an cnvironmcntal growth cabinet (Fi-totron 6OOL1) at a constant tcmpcraturc (IS‘C) at 100% humidity under the same lighting rcgimc. Rycgrass plunts wcrc grown in soil for 3 weeks before rccciving a single I.85 MBq pulse of “C-CO1, with a 2 h assimilation period. Two days before pulse labclling. root-soil and shoot chambers were partitioned with an air-tight seal. The experiment consisted of four treatments: (I) aerobic control; (2) root-soil chamber anaerobic for 5 h before pulse labelling: (3) root-soil chamber anaerobic for IO h with the pulse label applied 5 h into the anaerobic period; (4) root-soil chamber anaerobic for 48 h with the pulse label applied 5 h into the anaerobic period. Acratcd root-soil chambers were turned anaerobic by continuously pumping NI into the chamber (Row rate 24 ml NZ min-‘) throughout the anaerobic period. Chambers wcrc rapidly reacrated by pumping air into them at a rate of 24 ml min-‘. Air and N, wcrc both bubbled through alkaline traps to remove rcspircd “C-CO:. Root-soil respiration rates were monitored daily for 7 days bcforc plants wcrc harvcstcd. Roots were rccovcred from the soil by carefully removing the roots from the nylon mesh tube in the plant growth chambers. Soil within the mesh was further examined for root fragments. which were removed with forceps. The roots wcrc washed gently in distilled water before analysis. Root and shoot “C activities wcrc then determined after drying at 55’C. The
C assimilation in plants under anaerobiosis Table
I. Airdrv
wcinhts
Treatment Total
plant
Shoot Root
for L. twrww Aerobic
wt (mg)
wt (mg) wt (mg)
Shoot, root Figures
ratio
represent
prawn
in prowth
Anaerobic
5h
kcut aerobic
or anaerobic
Anaerobx
10 h
58 h
51 +-6
70 * 7
58 2 6
53 t_ I3
35 2 5
4a* to
35 + 5
39 * 7
17 = 2
22 f
132
2.2 5 0.4 the means
5
3.0 c 1.3
of 4 microcosms
Oven dry soil was used to determine “C activity of the soil. Root. shoot and soil (both inside and outside the mesh tube) lJC activities were determined by wet digestion with a chromic acid mixture with trapping of CO: evolved in a 5 ml I M NaOH trap (Dalal, 1979). The lJC activity of these traps and those used to trap respired lJC were determined by liquid scintillation counting. Pica-Fluor 40 (United Technologies Packard) was used as the scintillant with a Minaxi Tri-Carb 4000 liquid scintillation counter. The lJC activities of the traps were determined against quenched standards. Unpaired r-tests were used to assess the significance of count data as variances precluded the use of a one-way analysis of variance. Daily root-soil “C respiration data were analysed using the Least Significant Interval (LSI) test, to assess the significance of the data obtnincd. RESULTS
There were no signihcant diffcrcnccs in air dry plant biomass (root, shoot and total) or in shoot-root mtios between any of the treatments (Table I), indicating that the short periods of anaerobiosis did not adversely all&t plant growth. The distribution of the pulse label within 30day old perennial ryegrass, when the roots were subjected to a number of anaerobic treatments is shown in Table 2. The plants subjected to IO and 48 h of anacrobiosis had significantly less (by IO and 50% respectively) label retained by the plant and lost from the roots into the soil than the aerobic controls. None of the anaerobic treatments differed significantly in terms of label retained by the plant and lost from the roots into the soil. It is suggested that the low value of “C recovered from plant and soil systems where plant growth chambers were anaerobic for 48 h, may of “C
in shoot
Acroblc
and
root-soil
Anaerobic
Treatment
3.2 + 0.6
2.7 2 0.3
be explained by high “‘CCO, shoot respiration, which was not determined in the experiment. The “C remaining in the plant shoots at harvesting and, therefore. 14C translocated below ground, was significantly different when comparing the aerobic control to the IO and 48 h anaerobic treatments (Table 2). Four times as much “C, in absolute terms was respired by the aerobic plants than by the plants that were anaerobic for 48 h. There were no significant differences in lJC remaining in the soil or in the plant roots at the end of the experiment. In the soil outside of the nylon mesh. lJC was undetectable. The distribution of C fixed by the plant expressed as a proportion of total fixed lJC (Table 2). shows that prolonging anaerobiosis. increased the “C translocated below ground. This increased ‘?Z translocated below ground is mainly respired by the root-soil and exuded into the soil. Three times as much of the total “C fixed by the plants was respired by the roots-soil for plants that were kept anaerobic for 48 h than by the aerobic controls, although the quantity of 14C retained by the plant and lost into the soil from the roots was only 30% of the control. For the shorter anucrobic periods. the proportion of ‘%Z respired by the root-soil was 1.2 (5 h) and I.5 (IO h) times as much as the aerobic control. The pattern of ‘Y respired by the root-soil was similar for the aerobic control plants as for plants kept anaerobic for 5 and IO h (Fig. 2. and Table 3). The longer period of anaerobiosis (48 h) significantly suppressed root-soil lJC respimtion during the first 24 h compared to the other anaerobic treatments. although it was not significantly different to the control. When the root-soil chambers were returned to aerobic conditions after 48 h, root-soil lJC respiration increased significantly (from day I to day 3). and then followed the pattern exhibited by the other treatments. Anaerobiosis increased “C remaining in the soil at harvest when expressed as a proportion of the total “C fixed by the plant (Table 2). For all anaerobic treatments, there was an increase in percentage “C remaining in the soil at harvest compared to aerobic controls. In
Analysis
2. Dlstnbution
IJ=1
I
(k SE of the mean).
soil recovered from each mesh tube was weighed and thoroughly mixed to ensure homogeneity for subsequent analysis.
Table
chamber
Anacroblc
5 h
(kRo
P’
ch;lmbcrs Anaerobic
of L. perrnnr IO h
Anaerobic
4X h
oven drv wcinht)
TOLlI
370.0 + 4x 0
159.1 + 59.2
151.7.
t 55.5
37.0..
+ II.1
Shoot
289.0 f
107.3 f 55.5
99.9’
t 44..t
11.1**
r 7.4
52.0
(78.0%) RWI
I4.U + 7.4 (4.0%)
RWI
so11 respiratwn
Figures
rcprcscni
unpaired There Figures
were
(30.0%) I.1 +o.n
37.0 f
from
44.4?
(3.0%) II.1
21.1 r 2.6
(19.0%)
(23.2%)
(29.396)
(57.0%)
a.1 + 3.0
7.4 + 0.7
3.3 f
aerobic
(4.9%)
(5.1%)
the means of 4 muxocosms
dificrcnccs
(0.7%) 3.7
2.2 c I.1 (O.ho/bl
Significant
(65.8%) I.1 * 0.4
I.5
(1.9%)
70.3 2 Ill.5
SO’1
(67.0%) 3.0 *
control
(+SE at
l.
of the mean).
5%.
l*.
I%
levels.
determined
using an
I-wst. no significant
in brackets
ditTcrcnces
arc count
data
bctwecn expressed
I.5
(9%)
anaerobic
treatments.
as a percentage
of net assimd?tcd
“C.
A. A. MEHARG and K. KILLHAM
0
t
2
4
3
5
6
was restored. However, root-soil respiration increased for anaerobic treatments compared to the control plants, This may be due to less efficient use of photoassimilates through glycolysis. The products of glycolysis are thought to relax cell membranes. leading to increased root exudation (Smucker. 1984). There may atso be a high energy cost, (depleting stored photoassimilates) in returning to normal metabolic function after anaerobiosis. The proportion of ‘*C remaining in the soil at harvest was high for all the anaerobic treatments compared to the aerobic control, suggesting that root exudation increases. Results of solution culture studies [e.g. Schumacher and Smucker (1985); Minchin and McNaughton f 1984): Wiedenroth and Poskuta (t98l)I show that root exudation increases under aerobic conditions. Ethanol (a major product of glycolysis) was identified in root exudates of peas under anaerobic conditions (Smucker and Erickson. 1976, 198-J), The longer periods of root anaerobiosis (48 h) initially inhibited root-soil respiration, providing additional evidence that translocation of photoassimilate to the roots is inhibited by anaerobiosis (Schumacher and Smuckcr, 1985: Nuritdinov and Vartapctian, 1976). This inhibition of C-tlow within ryegrass was confumcd by a parallel experiment with plants of the same stage of development and grown under nctfr identical conditions (the only real differcncc being that the plants were not grown within the nylon tubes). Unc sot of plants was kept anaerobic for 48 h and then the plants wcrc harvcstcd. Of the total “C tixcd under thcsc conditions. only I I% wits transtocatcd below ground, with 10.2% b&g respired by the routs-soit. For the aerobic control, 39% of the total “C fixed was translocatcd below ground with 22.8% of this budget being rcspircd by the roots/soil. In our experiment, 57% of the total “C retained by plants anaerobic for 48 h was respired in the next 7 days. Only 3% of the ‘*C retained. remained in the roots at harvest. For the aerobic control, 19% of the ‘%.Iretained was respired by the roots-soil and 4% remained in the root. With a long exposure by the roots to anaerobic conditions, there may be a high energy cost (in terms of stored photoassimilates) in returning to normal metabolic function. It is suggested that increased translocation of “C into the root on cessation of anaerobiosis may explain the large percentage of 14C (57%) trandocatcd below ground when the plants were anaerobic for 48 h. A long post-anaerobiosis period of 7 days was rcquircd to fully assess the distribution of fixed lal~l within the plant.
7
DOYS
Fig. 2. Daify
root-soil
“C-CC+
respiration
for
aerobic
control plants (0) and for plants subjected to 5 h (U). 10 h (0) and 48 h (0) anaerobic soil conditions; expressed as
kBq g-’ day-’
air dry plant
weight,
terms of actual “C (Bq g-t air dry plant weight), however. for the control, 5 and IO h anaerobic periods, the activities remaining in the soil were simitar. The longer period of anacrobiosis dccreascd by half the actual rJC remaining in the soil.
Our results show that when roots of pcrcnnial rycgrass wcrc subjcctcd to short-term anacrobiosis, the label rctaincd by the plant and rclcascd into the soil was significantly less for the anaerobic treatments than for the control plants. Wiedcnroth and Poskuta (1981) found thnt apparent photosynthesis of wheat seedlings decreased with increasing exposure of roots (in solution culture) to anacrobiasis. They found that during the first 48 h of anaerobiosis. apparent photosynthesis differed fittlc from the aerobic control. Only more cxtcndcd periods of anaerobiosis seriously altcrcd apparent photosynthesis. It would appear from our study that inhibition of photoassimilation is much more rapid for perennial ryegrass. High shoot respiration may have accounted for the decreased ‘% retained by the plants in the anaerobic treatments. The non-significant reduction of root-soil rcspiration of rycgrass roots subjected to anaerobiosis suggests that the plant quickly returned to normal metabolism when an aerobic root environment fable
3.
LC~SIsignific;rnl
inlcrv&
rcspirition Aerobic Treatmcnr
calculated data
shown
Anaerobic jkBq
DCI,‘~ I
26.6
2 3 4 5 6 7
13.3 5.2 5.5 I.1 1.1 0.4
4.4 5.5 4.i 0.7 1.1 0.4 0.4
5h g-’
for
daily
in Fig.
root+oil
“C-CO:
2
Anaerobic
10”
An;lcrabic
day “)
i4.4 5.5 4.1 1.i I.5 0.7 0.7
22 9.2 1.1 2.6 2.2 2.2 2.2
JX h
C assimilation
in plamts under anaerobiosis
The percentage of total fixed “C respired by the roots-soil in the experiment reported here for the aerobic control plants corresponds well with the range 18-38% of ‘*C respired by the roots-soil of perennial ryegrass of the same stage of development. grown in a range of soils of varying pH (Meharg and Killham. 1990). Lambers (1987) reviewed the literature on the fate of C translocated to roots and estimated that root respiration accounted for I’_-29% of total photoassimilated C. The amount of “C remaining in the soil on harvesting of pulse labelled perennial ryegrass showed that 0.7% of total 14C fixed remained in the soil after 8 days (Meharg and Killham, 1988) which compares well with the 0.6% reported here. In conclusion, short periods of anaerobiosis, reduced the retention of assimilated C and increased the proportional loss of retained C due to root-soil respiration and root exudation. A longer period of anacrobiosis had a more dramatic effect on the distribution of the label, initially suppressing the translocation of label below ground, and then causing an incrcascd loss of label from root-soil respiration after the removal of anaerobic conditions. cl~lmo~~k~c~~c~~~~,~~r--Wc wish to acknowledge the Departmcnt of Agriculture for Northern Ireland for the studcntship to hndrcw Mcharg which cnablcd this study to bc undcrtakcn.
Rowcn G. D. (IYXO) Misconceptions. concepts and approaches in rhilosphcrc biology. In Conremporaq ~lic~rohiol Ec&xy (D. C. Ellwood. J. N. tlcdgcr. M. L. Latham. J. M. Lynch and J. 11. Slatcr. Eds), pp. 2X3 .30-I. Academic Press, London. Crawford R. M. M. (lY78) Metabolic adaptations to anoxia. In Pl~mr Li/i, in Am~crohic Dwironnr~vrs (D. D. llook and R. M. M. Crawford. Eds). .nn. . II9 136. Ann Arbor Science. Michigan. Dalal R. C. (IY79) Simple procedure for the dctcrmination of total carbon and its radioactivity in soils and plant malcrials. Amr/y.vr 104, 151-154.
647
Jenkinson D. S. and Powlson D. S. (1976) The effects of bitidal treatments on metabolism in soil-V. A method for measuring soil biomass. Soil Biology & Biochemicrry 8, 209-213. Lambers H. (1987) Growth. respiration, exudation and symbiotic associations: the fate of carbon translocated to the roots. In Roof Developmenr and Function (P. J. Gregory, J. V. Lake and D. A. Rose, Eds). pp. 125-146. Cambridge University Press. London. Meharg A. A. and Killham K. (1988) Comparison of carbon flow from pre-labelled and pulse labelled plants. Planr and Soil 112, 225-231. Meharg A. A. and Killham K. (1990) The effect of soil pH on rhizosphere carbon flow of Lolium perenne. Plant und Soil (in press). Minchin P. E. H. and McNaughton G. S. (1984) Exudation of recently fixed carbon by non-sterile roots. Journul of Experimental Boruny 35. 7482. Nuritdinov N. and Vartapetian B. B. (1976) Transport of “C-sucrose in cotton plants under conditions of root oxygen deficiency. Doklady LJSSR Academy Sciences 228, 149. Schumacher T. E. and Smucker A. J. M. (1985) Carbon transport and root respiration of split root systems of Phureolus culgaris subjected to short term anonia. Plunr Physiology 78. 359-364. Smucker A. J. M. (1984) Carbon utilization and losses by plant root systems. In Roars. Nutrient und Water Injlux and Plunf Growrh (S. A. Barber and D. R. Bouldin. Eds). pp. 27-46. ASA SpcTial Publication No. 49. Madison. Smuckcr A. J. M. and Erickson A. E. (1976) An aseptic mist chamber system: a method for measuring root proccsscs of peas. rlaronomy Journul 68. 59 62. Smucker A. J. M. and Erickson A. E. (1987) Anaerobic stimulation of root exudates and discasc of peas, Pkunr and Soil 99. 423 433. Vartapetian B. 8.. Andrecva I. N. and Nuritdinov N. (lY7X) Plant cells under oxygen stress. In Pkunf Lift in Anuerohic Em~irunmc*nfs (D. D. tlook and R. M. M. Crawford, Eds). pp. I3 88. Ann Arbor Science, Michigan. Wicdenroth E. and Poskuta 1. (1981) The influence of oxygen dclicicncy in roots on CO, exchange rates of shoots and distribution of “C-photoassimilates of wheat seedlings. Zrirschrij Pflun:enpi,ysioiu~i~ Boclfnkunde 103, 459 -467.
1990
W38-0717.90 S3.00 + 0.00 Copyright C 1990 Pcrgamon Press plc
CARBON DISTRIBUTION WITHIN THE PLANT AND RHIZOSPHERE FOR LOLIUM PERENNE SUBJECTED TO ANAEROBIC SOIL CONDITIONS A. A. MEHARG* Department
of Soil Science. The University
and K. KlLLHAMt
of Aberdeen.
(Accep~prrJ I5 Junuury
Meston
Walk,
Aberdeen
AB9 ZUE.
Scotland
1990)
Summary-Perennial ryegrass was subjected to a range of anaerobic treatments. The distribution of C within the plant was determined by pulse labclling the shoots with “C
INTRODUCTION
0: tension around plant roots causes many complex chanpcs in plant mctnbolic proccsscs and damages root cell structure. Anaerobic mctaholism of photoassimilatcs is much less ctficicnt in terms of cncrgy mol *’ substrate than aerobic mctaholism (Smuckcr. 1984). leading to C starvation within the plant roots (Vartapctian et nl., 1978). When plant roots arc placed in an anaerobic cnvironmcnt the trunslocation of C from shoots to roots is inhibited (Nuritdinov and Vartapctian, 1976). which cxaccrbates C starvation. Anaerobic metabolism in plant roots products (through glycolysis) products such as ethanol. acctaldchydc and lactate. Thcsc compounds arc toxic to plant roots and arc thought to relax ccl1 mcmbrancs, thus increasing the loss of C from plant roots into the rhizosphcrc. Thcsc compounds released may provide substrates for pdthogcnic micro-organisms, which further damage the plant (Crawford, 1978; Smuckcr and Erickson. 1976. 1987). Studies to invcstigatc C distribution in plants where the roots have been subjected to anaerobic stress have been carried out in solution culture [e.g. Schumacher and Smuckcr (1985); Minchin and McNaughton (1984); Wicdcnroth and Poskuta (1981); Nuritdinov and Vartapctian (1976)] or in aseptic mist chambers [e.g. Smuckcr and Erickson (1976. 1987)j. Relating solution culture studies of the rhizosphcrc to soil-grown plants has usually littlc or no rclcvancc (Bowen, 1980). Our purpose was to study the photoassimilation and distribution of a “C-CO: pulse label fixed by pcrcnnial rycgrass grown Rcduccd
‘Present address: Department of Biological Sciences. University of Exeter. Hatherly Laboratories. Prince of Wales Road. Exeter EX4 4P.S. England. tTo whom all correspondence should be addressed.
in soil, whcrc the roots of the plant were subjcctcd to a range of anaerobic trcatmcnts. MATERIAIS
ANI) hlETIIOI)S
Soil
The soil used in the cxpcrimcnt was a sandy loam (Tarvcs scrics) Tillycorthic, N.E. Scotland, sampled to a depth of 2Ocm. Some characteristics of the soil were: 3.7% organic C [dctcrmincd by wet digestion, Dalal (1979)j; 256 mg biomass C kg-’ o.d. soil using the fumigation-incubation method of Jcnkinson and Powlson (1976) and pH 6.3 (using a 2.5: I H,O-soil slurry). Plunt
luhdling
and monitoring
chunkhrrs
A plant growth chamber (Fig. I) was designed in which grass plants could bc labellcd using “C-CO1. with subsequent monitoring of root-soil respiration. The base (root-soil chamber) was a section of PVC tubing (length, 21 cm; o.d., 8.3 cm; i.d., 7.3 cm) which was capped at one end and had three ports on one side at 5cm spacings. Glass tubes were inserted through holes bored in No. 20 silicone suba seals in the top and bottom ports. The central port was fitted with a No. 20 silicone suba seal. A PVC disc with a hole in the centrc, through which a section of a 5 ml syringe barrel was placed, was sealed into position just below the top of the base of the monitoring chamber. using high vacuum silicone grease. The disc was scaled into position before pulse labclling of the plants. The shoot chamber was a length of clear perspcx tubing (length, 28 cm; o.d., 8.3 cm; i.d.. 7 cm), capped at one end. fitted with plastic nozzles, which served as air inlets or outlets. The top section was lathed at its base to fit tightly onto the base section (Fig. I) and scaled with high vacuum silicone grease.
A. A. MEHARO and K. KILLHAM growth chambers were checked to ensure that there were no leaks either externally, or between the root-soil and shoot chambers. This was done by pumping air into the root-soil chamber at IOOml min-’ and air outlets of both root-soil and shoot chambers monitored to ensure no air was escaping into the shoot chamber. Plant culture
Fig. I. Design of plant-soil growth chambers. (I) Soil-root chamber; (2) shoot chamber; (2) partition between shoot and soil-root chamber: (4) air from pump; (5) suba-scaled watcrinp port; (6) air to alkaline trap: (7) air from pump; (4) air to alkaline trap: (9) plant joint: (IO) lathed port.
The “C-CO2 was introduccd to the shoot chamber using a suba scaled. quick-tit flask conncctcd by a side-arm to the shoot chamber. Labcllcd CO2 was gcncratcd by injecting “C-labcllcd Na,CO, into concontrated lactic acid. After sufhcicnt time (2 h) for plants to assimilate tho label. ambient air was pumped continuously through the shoot chamber, and the “C-CO? being removed by bubbling exhaust gases through 5 ml I M NaOH in a IO ml test tube. The NaOH traps were changed daily. To monitor root-soil respiration, shoot and root-soil chambers wcrc isolated using a PVC disc with thr grass planted through the syringe barrel. The shoots and root-soil were separated from each other using high vacuum silicone grease around the base of the grass shoots and between the shoots and the sides of the syringe barrel. Moist air was then pumped continuously into the plant growth chamber through both shoot and root-soil chambers at a flow rate of 24 ml min-‘. and respired “C-CO, trapped in 5 ml I M NaOH in a IO ml test tube. Air pumped through the shoot chamber was passed through the alkaline trap to prevent the release of the label into the atmosphere. Shoot respiration data wcrc not reported as shoot rcspircd and non-assimilated label could not be partitioned in the early stage of the cxpcrimcnts. Moisture content of the soil used in this cxpcrimcnt was initially a ficld capacity. At harvest, soil water potential was -2.8 kPa. Thcsc water potentials would be expcricnccd by plants growing under field conditions. Although continually pumping of the root-soil chamber with air will have contributed to the loss of soil moisture the soil water potential data suggested the plants wcrc not subjected to advcrsc soil conditions (0 to -2.8 kPa). Air inlet tubes were fitted with sterilized mcmbranc tiltcr units. The plant
Lo&m perenne (Preference) was grown for 5 days in sand irrigated with a quarter-strength Hoaglands nutrient solution. following germination (4 days) in glass Petri dishes. lined vvith damp filter paper. The ryegrass plants were grown under a light bank of 8 x 85 watt fluorescent tubes (light intensity 20,000 lux) with a I2 h photoperiod, with a maximum day-time temperature of 25°C and a minimum nighttime temperature of ZO’C. Randomly selected plants (26 mg air dry weight) were then transferred to 53 pm nylon mesh tubes containing loosely-packed moist soil (moisture content 35.7%. soil water potential -0.49 kPa). and planted at a density of 5 plants per tube. Nylon mesh tubes were constructed from 6 by 20 cm rectangles of the mesh folded in half and the long edge and one of the short cdgcs heat scaled using a hot soldering iron. The tubes wcrc then placed in the root-soil chamber and soil (950 p. of the same moisture content as that used in the tubes) was packed around the tube to hold it in a central position and the PVC disc placed in position on the top of the soil. The plants were then grown in an cnvironmcntal growth cabinet (Fi-totron 6OOL1) at a constant tcmpcraturc (IS‘C) at 100% humidity under the same lighting rcgimc. Rycgrass plunts wcrc grown in soil for 3 weeks before rccciving a single I.85 MBq pulse of “C-CO1, with a 2 h assimilation period. Two days before pulse labclling. root-soil and shoot chambers were partitioned with an air-tight seal. The experiment consisted of four treatments: (I) aerobic control; (2) root-soil chamber anaerobic for 5 h before pulse labelling: (3) root-soil chamber anaerobic for IO h with the pulse label applied 5 h into the anaerobic period; (4) root-soil chamber anaerobic for 48 h with the pulse label applied 5 h into the anaerobic period. Acratcd root-soil chambers were turned anaerobic by continuously pumping NI into the chamber (Row rate 24 ml NZ min-‘) throughout the anaerobic period. Chambers wcrc rapidly reacrated by pumping air into them at a rate of 24 ml min-‘. Air and N, wcrc both bubbled through alkaline traps to remove rcspircd “C-CO:. Root-soil respiration rates were monitored daily for 7 days bcforc plants wcrc harvcstcd. Roots were rccovcred from the soil by carefully removing the roots from the nylon mesh tube in the plant growth chambers. Soil within the mesh was further examined for root fragments. which were removed with forceps. The roots wcrc washed gently in distilled water before analysis. Root and shoot “C activities wcrc then determined after drying at 55’C. The
C assimilation in plants under anaerobiosis Table
I. Airdrv
wcinhts
Treatment Total
plant
Shoot Root
for L. twrww Aerobic
wt (mg)
wt (mg) wt (mg)
Shoot, root Figures
ratio
represent
prawn
in prowth
Anaerobic
5h
kcut aerobic
or anaerobic
Anaerobx
10 h
58 h
51 +-6
70 * 7
58 2 6
53 t_ I3
35 2 5
4a* to
35 + 5
39 * 7
17 = 2
22 f
132
2.2 5 0.4 the means
5
3.0 c 1.3
of 4 microcosms
Oven dry soil was used to determine “C activity of the soil. Root. shoot and soil (both inside and outside the mesh tube) lJC activities were determined by wet digestion with a chromic acid mixture with trapping of CO: evolved in a 5 ml I M NaOH trap (Dalal, 1979). The lJC activity of these traps and those used to trap respired lJC were determined by liquid scintillation counting. Pica-Fluor 40 (United Technologies Packard) was used as the scintillant with a Minaxi Tri-Carb 4000 liquid scintillation counter. The lJC activities of the traps were determined against quenched standards. Unpaired r-tests were used to assess the significance of count data as variances precluded the use of a one-way analysis of variance. Daily root-soil “C respiration data were analysed using the Least Significant Interval (LSI) test, to assess the significance of the data obtnincd. RESULTS
There were no signihcant diffcrcnccs in air dry plant biomass (root, shoot and total) or in shoot-root mtios between any of the treatments (Table I), indicating that the short periods of anaerobiosis did not adversely all&t plant growth. The distribution of the pulse label within 30day old perennial ryegrass, when the roots were subjected to a number of anaerobic treatments is shown in Table 2. The plants subjected to IO and 48 h of anacrobiosis had significantly less (by IO and 50% respectively) label retained by the plant and lost from the roots into the soil than the aerobic controls. None of the anaerobic treatments differed significantly in terms of label retained by the plant and lost from the roots into the soil. It is suggested that the low value of “C recovered from plant and soil systems where plant growth chambers were anaerobic for 48 h, may of “C
in shoot
Acroblc
and
root-soil
Anaerobic
Treatment
3.2 + 0.6
2.7 2 0.3
be explained by high “‘CCO, shoot respiration, which was not determined in the experiment. The “C remaining in the plant shoots at harvesting and, therefore. 14C translocated below ground, was significantly different when comparing the aerobic control to the IO and 48 h anaerobic treatments (Table 2). Four times as much “C, in absolute terms was respired by the aerobic plants than by the plants that were anaerobic for 48 h. There were no significant differences in lJC remaining in the soil or in the plant roots at the end of the experiment. In the soil outside of the nylon mesh. lJC was undetectable. The distribution of C fixed by the plant expressed as a proportion of total fixed lJC (Table 2). shows that prolonging anaerobiosis. increased the “C translocated below ground. This increased ‘?Z translocated below ground is mainly respired by the root-soil and exuded into the soil. Three times as much of the total “C fixed by the plants was respired by the roots-soil for plants that were kept anaerobic for 48 h than by the aerobic controls, although the quantity of 14C retained by the plant and lost into the soil from the roots was only 30% of the control. For the shorter anucrobic periods. the proportion of ‘%Z respired by the root-soil was 1.2 (5 h) and I.5 (IO h) times as much as the aerobic control. The pattern of ‘Y respired by the root-soil was similar for the aerobic control plants as for plants kept anaerobic for 5 and IO h (Fig. 2. and Table 3). The longer period of anaerobiosis (48 h) significantly suppressed root-soil lJC respimtion during the first 24 h compared to the other anaerobic treatments. although it was not significantly different to the control. When the root-soil chambers were returned to aerobic conditions after 48 h, root-soil lJC respiration increased significantly (from day I to day 3). and then followed the pattern exhibited by the other treatments. Anaerobiosis increased “C remaining in the soil at harvest when expressed as a proportion of the total “C fixed by the plant (Table 2). For all anaerobic treatments, there was an increase in percentage “C remaining in the soil at harvest compared to aerobic controls. In
Analysis
2. Dlstnbution
IJ=1
I
(k SE of the mean).
soil recovered from each mesh tube was weighed and thoroughly mixed to ensure homogeneity for subsequent analysis.
Table
chamber
Anacroblc
5 h
(kRo
P’
ch;lmbcrs Anaerobic
of L. perrnnr IO h
Anaerobic
4X h
oven drv wcinht)
TOLlI
370.0 + 4x 0
159.1 + 59.2
151.7.
t 55.5
37.0..
+ II.1
Shoot
289.0 f
107.3 f 55.5
99.9’
t 44..t
11.1**
r 7.4
52.0
(78.0%) RWI
I4.U + 7.4 (4.0%)
RWI
so11 respiratwn
Figures
rcprcscni
unpaired There Figures
were
(30.0%) I.1 +o.n
37.0 f
from
44.4?
(3.0%) II.1
21.1 r 2.6
(19.0%)
(23.2%)
(29.396)
(57.0%)
a.1 + 3.0
7.4 + 0.7
3.3 f
aerobic
(4.9%)
(5.1%)
the means of 4 muxocosms
dificrcnccs
(0.7%) 3.7
2.2 c I.1 (O.ho/bl
Significant
(65.8%) I.1 * 0.4
I.5
(1.9%)
70.3 2 Ill.5
SO’1
(67.0%) 3.0 *
control
(+SE at
l.
of the mean).
5%.
l*.
I%
levels.
determined
using an
I-wst. no significant
in brackets
ditTcrcnces
arc count
data
bctwecn expressed
I.5
(9%)
anaerobic
treatments.
as a percentage
of net assimd?tcd
“C.
A. A. MEHARG and K. KILLHAM
0
t
2
4
3
5
6
was restored. However, root-soil respiration increased for anaerobic treatments compared to the control plants, This may be due to less efficient use of photoassimilates through glycolysis. The products of glycolysis are thought to relax cell membranes. leading to increased root exudation (Smucker. 1984). There may atso be a high energy cost, (depleting stored photoassimilates) in returning to normal metabolic function after anaerobiosis. The proportion of ‘*C remaining in the soil at harvest was high for all the anaerobic treatments compared to the aerobic control, suggesting that root exudation increases. Results of solution culture studies [e.g. Schumacher and Smucker (1985); Minchin and McNaughton f 1984): Wiedenroth and Poskuta (t98l)I show that root exudation increases under aerobic conditions. Ethanol (a major product of glycolysis) was identified in root exudates of peas under anaerobic conditions (Smucker and Erickson. 1976, 198-J), The longer periods of root anaerobiosis (48 h) initially inhibited root-soil respiration, providing additional evidence that translocation of photoassimilate to the roots is inhibited by anaerobiosis (Schumacher and Smuckcr, 1985: Nuritdinov and Vartapctian, 1976). This inhibition of C-tlow within ryegrass was confumcd by a parallel experiment with plants of the same stage of development and grown under nctfr identical conditions (the only real differcncc being that the plants were not grown within the nylon tubes). Unc sot of plants was kept anaerobic for 48 h and then the plants wcrc harvcstcd. Of the total “C tixcd under thcsc conditions. only I I% wits transtocatcd below ground, with 10.2% b&g respired by the routs-soit. For the aerobic control, 39% of the total “C fixed was translocatcd below ground with 22.8% of this budget being rcspircd by the roots/soil. In our experiment, 57% of the total “C retained by plants anaerobic for 48 h was respired in the next 7 days. Only 3% of the ‘*C retained. remained in the roots at harvest. For the aerobic control, 19% of the ‘%.Iretained was respired by the roots-soil and 4% remained in the root. With a long exposure by the roots to anaerobic conditions, there may be a high energy cost (in terms of stored photoassimilates) in returning to normal metabolic function. It is suggested that increased translocation of “C into the root on cessation of anaerobiosis may explain the large percentage of 14C (57%) trandocatcd below ground when the plants were anaerobic for 48 h. A long post-anaerobiosis period of 7 days was rcquircd to fully assess the distribution of fixed lal~l within the plant.
7
DOYS
Fig. 2. Daify
root-soil
“C-CC+
respiration
for
aerobic
control plants (0) and for plants subjected to 5 h (U). 10 h (0) and 48 h (0) anaerobic soil conditions; expressed as
kBq g-’ day-’
air dry plant
weight,
terms of actual “C (Bq g-t air dry plant weight), however. for the control, 5 and IO h anaerobic periods, the activities remaining in the soil were simitar. The longer period of anacrobiosis dccreascd by half the actual rJC remaining in the soil.
Our results show that when roots of pcrcnnial rycgrass wcrc subjcctcd to short-term anacrobiosis, the label rctaincd by the plant and rclcascd into the soil was significantly less for the anaerobic treatments than for the control plants. Wiedcnroth and Poskuta (1981) found thnt apparent photosynthesis of wheat seedlings decreased with increasing exposure of roots (in solution culture) to anacrobiasis. They found that during the first 48 h of anaerobiosis. apparent photosynthesis differed fittlc from the aerobic control. Only more cxtcndcd periods of anaerobiosis seriously altcrcd apparent photosynthesis. It would appear from our study that inhibition of photoassimilation is much more rapid for perennial ryegrass. High shoot respiration may have accounted for the decreased ‘% retained by the plants in the anaerobic treatments. The non-significant reduction of root-soil rcspiration of rycgrass roots subjected to anaerobiosis suggests that the plant quickly returned to normal metabolism when an aerobic root environment fable
3.
LC~SIsignific;rnl
inlcrv&
rcspirition Aerobic Treatmcnr
calculated data
shown
Anaerobic jkBq
DCI,‘~ I
26.6
2 3 4 5 6 7
13.3 5.2 5.5 I.1 1.1 0.4
4.4 5.5 4.i 0.7 1.1 0.4 0.4
5h g-’
for
daily
in Fig.
root+oil
“C-CO:
2
Anaerobic
10”
An;lcrabic
day “)
i4.4 5.5 4.1 1.i I.5 0.7 0.7
22 9.2 1.1 2.6 2.2 2.2 2.2
JX h
C assimilation
in plamts under anaerobiosis
The percentage of total fixed “C respired by the roots-soil in the experiment reported here for the aerobic control plants corresponds well with the range 18-38% of ‘*C respired by the roots-soil of perennial ryegrass of the same stage of development. grown in a range of soils of varying pH (Meharg and Killham. 1990). Lambers (1987) reviewed the literature on the fate of C translocated to roots and estimated that root respiration accounted for I’_-29% of total photoassimilated C. The amount of “C remaining in the soil on harvesting of pulse labelled perennial ryegrass showed that 0.7% of total 14C fixed remained in the soil after 8 days (Meharg and Killham, 1988) which compares well with the 0.6% reported here. In conclusion, short periods of anaerobiosis, reduced the retention of assimilated C and increased the proportional loss of retained C due to root-soil respiration and root exudation. A longer period of anacrobiosis had a more dramatic effect on the distribution of the label, initially suppressing the translocation of label below ground, and then causing an incrcascd loss of label from root-soil respiration after the removal of anaerobic conditions. cl~lmo~~k~c~~c~~~~,~~r--Wc wish to acknowledge the Departmcnt of Agriculture for Northern Ireland for the studcntship to hndrcw Mcharg which cnablcd this study to bc undcrtakcn.
Rowcn G. D. (IYXO) Misconceptions. concepts and approaches in rhilosphcrc biology. In Conremporaq ~lic~rohiol Ec&xy (D. C. Ellwood. J. N. tlcdgcr. M. L. Latham. J. M. Lynch and J. 11. Slatcr. Eds), pp. 2X3 .30-I. Academic Press, London. Crawford R. M. M. (lY78) Metabolic adaptations to anoxia. In Pl~mr Li/i, in Am~crohic Dwironnr~vrs (D. D. llook and R. M. M. Crawford. Eds). .nn. . II9 136. Ann Arbor Science. Michigan. Dalal R. C. (IY79) Simple procedure for the dctcrmination of total carbon and its radioactivity in soils and plant malcrials. Amr/y.vr 104, 151-154.
647
Jenkinson D. S. and Powlson D. S. (1976) The effects of bitidal treatments on metabolism in soil-V. A method for measuring soil biomass. Soil Biology & Biochemicrry 8, 209-213. Lambers H. (1987) Growth. respiration, exudation and symbiotic associations: the fate of carbon translocated to the roots. In Roof Developmenr and Function (P. J. Gregory, J. V. Lake and D. A. Rose, Eds). pp. 125-146. Cambridge University Press. London. Meharg A. A. and Killham K. (1988) Comparison of carbon flow from pre-labelled and pulse labelled plants. Planr and Soil 112, 225-231. Meharg A. A. and Killham K. (1990) The effect of soil pH on rhizosphere carbon flow of Lolium perenne. Plant und Soil (in press). Minchin P. E. H. and McNaughton G. S. (1984) Exudation of recently fixed carbon by non-sterile roots. Journul of Experimental Boruny 35. 7482. Nuritdinov N. and Vartapetian B. B. (1976) Transport of “C-sucrose in cotton plants under conditions of root oxygen deficiency. Doklady LJSSR Academy Sciences 228, 149. Schumacher T. E. and Smucker A. J. M. (1985) Carbon transport and root respiration of split root systems of Phureolus culgaris subjected to short term anonia. Plunr Physiology 78. 359-364. Smucker A. J. M. (1984) Carbon utilization and losses by plant root systems. In Roars. Nutrient und Water Injlux and Plunf Growrh (S. A. Barber and D. R. Bouldin. Eds). pp. 27-46. ASA SpcTial Publication No. 49. Madison. Smuckcr A. J. M. and Erickson A. E. (1976) An aseptic mist chamber system: a method for measuring root proccsscs of peas. rlaronomy Journul 68. 59 62. Smucker A. J. M. and Erickson A. E. (1987) Anaerobic stimulation of root exudates and discasc of peas, Pkunr and Soil 99. 423 433. Vartapetian B. 8.. Andrecva I. N. and Nuritdinov N. (lY7X) Plant cells under oxygen stress. In Pkunf Lift in Anuerohic Em~irunmc*nfs (D. D. tlook and R. M. M. Crawford, Eds). pp. I3 88. Ann Arbor Science, Michigan. Wicdenroth E. and Poskuta 1. (1981) The influence of oxygen dclicicncy in roots on CO, exchange rates of shoots and distribution of “C-photoassimilates of wheat seedlings. Zrirschrij Pflun:enpi,ysioiu~i~ Boclfnkunde 103, 459 -467.