The effect of potassium deficiency on growth and N2-fixation in Trifolium repens
Henning H?gh-Jensen
Department of Agricultural Sciences,Royal Veterinary and Agricultural University,H?jbakkegaard Alle′13,DK-2630Taastrup, Denmark
e-mail:hhj@kvl.dk
Received7January2003;revised31March2003
The effects of potassium(K)deficiency on growth,N2-fixation and photosynthesis in white clover(Trifolium repens L.)were investigated using natural occurring gas fluxes on the nodules in real time of plants under three contrasting relative addition rates of K causing mild K deficiency,or following abrupt withdrawal of the K supply causing strong K deficiency of less than0.65%in dry matter.A steady-state below-optimum K supply rate led to an increase in CO2-fixation per unit leaf surface area as well as per plant leaf surface.However, nitrogenase activity per unit root weight and per unit nodule weight was maintained,as was the efficiency with which electrons were allocated to the reduction of N2in the nodules. Abrupt K removals stimulated nodule growth strongly without delay,but as K concentrations decreased in the plant tissue a significant decline in nitrogenase activity per unit root weight as well as
per unit nodule mass occurred.Further,the rate of
photosynthesis per unit leaf area was unaffected,while the
CO2acquisition for the plant as a whole increased due to an
expansion of total leaf area whereas the leaf area per unit leaf
weight was unaffected.The ratio between CO2-fixation and
N2-fixation increased,although not statistically significant,
under short-term K deprivation as well as under long-term
low K supply indicating a downregulation of nodule activity
following morphological and growth adjustments.This down-
regulation took place despite a partly substitution of the K by
Na.It is concluded that N2-fixation does not limit the growth
of K-deprived clover plants.K deprivation induces changes in
the relative growth of roots,nodules,and shoots rather than
changes in N and/or carbon uptake rates per unit mass or area
of these organs.
Introduction
Legumes are important in agriculture due to their ability to reduce atmospheric N2symbiotically within their root nodules,thus making it available for plants.It is well docu-mented that legume growth and nitrogen(N)accumulation are extraordinarily responsive to K supply(Blaser and Brady 1950,Drysdale1965,Duke and Collins1985,Mengel and Steffens1985).The impact of K deficiency on N2fixation is, however,not well understood.
Generally,plant responses to nutrient deficiencies follow the same pattern,involving compensatory changes in alloca-tion,to maximize acquisition of those resources that most directly limit growth(Bloom et al.1985).On the other hand, compensatory resource allocations cannot readily ameliorate stresses that have direct damaging effects on plants.In the short term,there may be considerable de-coupling between plant nutrient uptake and plant growth as it may take some time before a specific nutrient concentration becomes limiting and before the downregulation of uptake of other nutrients or photosynthesis start to act,i.e.there will be a lag-time. However,if a steady-state situation can be obtained in nutrient availability,plants are expected to allocate resources preferentially to the functions limiting growth most strongly (Bloom et al.1985,Chapin et al.1987,H?gh-Jensen and Pedersen2003).
Under environmental stress,most N2-fixing legumes are capable of maintaining a high metabolic activity in their root nodules.The response of N2-fixation to envir-onmental stress is species specific and the response will
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Abbreviations–Ar-ID,argon-induced decline;EAC,electron allocation efficiency;K,potassium;Na,sodium;RAR,relative addition rate; SLA,specific leaf area.
440Physiol.Plant.119,2003
reflect the duration and severity of the stress,the plant growth history and will be complicated by effects on nodulation and anatomical adaptation in the longer term(Walsh1995).Nitrogenase activity is particularly sensitive to abiotic stresses such as salinity(Serraj et al. 1994)or drought(Durand et al.1987),although phosphorus supply does not limit nitrogenase activity (H?gh-Jensen et al.2002).
Present understanding of the effect of K supply on legume growth and N2-fixation is based on experiments adding15N2gas to the growth compartment(Mengel et al.1974),using acetylene reduction techniques(Duke et al.1980,Collins and Duke1981,Barta1982,Collins and Lang1985),or by adding15N to the growth media (Feigenbaum and Mengel1979,Sangakkara et al.1996). These methodological approaches have lead to the view that K supply does affect the plant yield but not the nitrogenase activity of the nodules.
To investigate this hypothesis,the objective of the present studies was to investigate the effects of K deficiency of N2-fixation,growth and morphology of white clover plants to K deficiency using naturally occurring gas fluxes on the nodules in real time.In order to obtain an integrated view of the effects of K deficiency,two experimental approaches were used.First,experiments that simulated long-term steady-state situations were conducted,using different relative addition rates of K(Ingestad1982,Ingestad and A gren1988).Secondly,the K supply was abruptly with-drawn and the plants were fully deprived of external K for 3weeks in order to study the short-term dynamic metabolic regulatory responses.
Materials and methods
Plant material and growth conditions
Seeds of white clover(Trifolium repens https://www.doczj.com/doc/fd18498008.html,kanova) were germinated in vermiculite.After1week,the plants were inoculated with a Rhizobium leguminosarum biovar trifolii strain(WPBS5of IGER,Aberystwyth,UK), which is known to lack uptake hydrogenase activity, thus enabling the measurement of H2evolution for nitrogenase activity.Seed germination and plant growth were carried out in a controlled environment chamber at 75%RH,with a16/8h day/night length,a20/15 C day/ night temperature,and a light intensity(PAR)of approximately300m mol photons mà2sà1at shoot level (Powerstar HQI-T400W/D,Osram,Germany).
While growing in the vermiculite medium,plants were watered twice a week with an N-free nutrient solution containing(mmol mà3):400CaCl2,200MgSO4,400 K2SO4,100NaH2PO4,50H3BO3,50FeC6H5O7,20 MnSO4,2ZnSO4,1Na2MoO4,0.5CuSO4,0.5NiSO4, and0.5CoCl3.Four weeks after planting,samples of six trifoliate plants were transplanted to4-l containers that were purged by ambient air through the solution with a flow-rate of approx.1l minà1.The plant samples were fixed in the lid by an inert plastic material(Terostat1, Henkel Surface Technologies,Pennsylvania,USA),posi-tioning most of the nodules above the nutrient solution. The solution was renewed twice a week until the experimen-tal period started and was then changed three times per week to maintain the nutrient content.After renewal,the biological buffer MES[2-(N-morpholino)-ethanesulphonic acid;10ml of a750-mmol mà3solution with pH6.0]was added to control the pH in the nutrient solution. Experimental protocols
The steady-state experiments were conducted after the white clover plants had adapted to three different growth rates controlled by relative addition rates(RAR),four containers per RAR,following the equation(Ingestad 1982,Ingestad and A gren1988):
Net daily K addition?Kett1TàKetT
?KetT?ee RAR:à1Teqn1 where K(t)is the K content per unit plant DW at time,t. Treatments started when plants were7weeks old(after sowing)and continued for22days.The RAR treatments of0.03,0.06and0.12g K gà1K dayà1were obtained by supplying the required amounts of K each morning(eqn1). Before adding K,the nutrient solution was sampled and kept frozen(à20 C)until analysis.After the plants had adapted to these treatments for22days,they were subjected to gas exchange measurements(completed within2days). After transfer from the100mmol mà3K concentration in the pre-experimental nutrient solution the plants adjusted to the new lower K supply of the RAR treat-ments by effluxing K during the first days after transfer. Assessed on the basis of the added amount of K and that amount remaining just before the next addition on the following day,it was evident that roots from plants in RAR0.03,0.06and0.12treatments showed a net efflux of K during the first2,3and10days,respectively. Thereafter,by the end of the24-h period between K-additions,plants at RAR0.03and0.06had depleted the K content of the nutrient solution entirely,whereas plants subjected to the0.12RAR treatment were not able to absorb all of the K supplied on a daily basis.The short-term experiment,which aimed at investigating the effects of an abrupt K deprivation,was conducted after growing plants with a non-limiting K supply(100mmol mà3)for 10weeks after sowing,after which the K supply was with-drawn from half of the plants.
For both groups of plants,controls and K-deprived, measurements started at the point of K withdrawal.Subse-quently,measurements were carried out every3–4days until the final sampling after22days.Four replicate containers, each with six clover plants,were sampled at random on each measurement day.
Shoot measurements
Shoot CO2and water vapour exchanges were measured using a differential CO2/H2O infrared gas analyser (Ciras-1,PP Systems,Herts,UK),recording the
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difference between the inlet and outlet concentration of the two gases.Each single container was placed in a25-l Perspex cuvette,subjected to a flow rate of30l air minà1. Chlorophyll fluorescence(FMS-1,Hansatech Instru-ments Ltd,Pentney,UK)was determined on three replicate leaves per pot over a period of6min following30min dark-adaptation.The youngest fully expanded leaves were selected and a light intensity equivalent to the ambient light of300m mol photons(PAR)mà2sà1was used.
Root measurements
All root gas exchange measurements were conducted using an open-flow system in conjunction with a flow-through H2analyser with a flow-rate of1000ml minà1 and a root volume of1200ml of which200ml contained a K-free nutrient solution into which the gas was bubbled.The lids,in which the plants were fixed,were transferred to the containers for gas exchange measure-ments and left to adjust for1h.The tightly fitting lid and the plastic material around the stem bases enclosed the root compartment effectively.Tightness was checked by a flow-rate meter on the outlet.CO2emission from nodulated roots was measured using a differential CO2/ H2O infrared gas analyser(Ciras-1).
Nitrogenase(EC1.7.99.2)activity was determined by measuring H2evolution in79:21(v/v)mixtures of N2:O2 and Ar:O2obtained by mixing high purity(99.999%) gases using mass-flow controllers.The electron allocation coefficient(EAC)is defined as1-(ANA/TNA),where ANA and TNA denote the apparent and total nitrogenase activ-ity,measured as H2evolution in the N2:O2and Ar:O2 mixture,respectively.ANA and the initial root respiration were measured for5min in the N2:O2gas mixture and then for35min in the Ar:O2mixture.TNA was determined as the peak H2evolution.Following the peak,Ar-ID was determined as the decline after30min.The EAC values obtained were between0.65and0.74,which corresponds with the near-optimum values reported by Hunt and Layzell (1993).N2-fixation is calculated as(TNA–ANA)/3.
The H2concentration in the dried gas stream was determined using a calibrated H2sensor(Qubit Systems Inc.,Kingston,Canada).The sensor was reconditioned every morning(Layzell et al.1989)by injecting2cm3 pure H2into the gas stream.
Tissue analysis
Following gas exchange measurements,roots were rinsed in deionized water,excised and blotted dry.FW of roots and shoots was determined.Nodules were removed from the root material and weighed.The shoot leaf area was measured(one-side)using a LI-3100Area Meter(LiCor Inc.,Lincoln,NE,USA).The plant material was frozen (à20 C),freeze-dried to constant weight and weighed again before being ground(mesh size of0.2mm). Element determination
Nitrogen in the ground plant material was analysed using an ANCA-SL Elemental Analyser coupled to a 20–20Tracermass Mass Spectrometer(Europa Scientific Ltd,Crewe,UK)using the Dumas combustion method. The remaining plant material was dry-ashed at550 C for 3h,solubilized in3M HCl,dried and solubilized again in1M HNO3before filtering.The plant material was dry ashed at550 C for3h;solubilized in3M HCl;dried and solubilized again in1M HNO3before filtering.In the plant samples and in samples taken from the nutrient solution in the experiment with the RAR approach,K and Na were determined by flame photometry(flame emission;Perkin-Elmer,Norwalk,CT,USA). Statistical methods
The data were analysed by regression analysis using the SAS ANOVA procedure(SAS Institute Inc.1993).Com-parison of the means for the individual treatments was done using a Waller-Duncan t-test.
Results
Steady-state responses to varying K supply rates
Total dry matter production was on average3.1g dry matter per plant and was not affected by the K supply rates(P?0.63).On average the plants had a relative growth rate of0.072dayà1on a dry matter basis in the experimental period.Neither shoots(P?0.44),roots (P?0.44)nor nodule(P?0.38)dry matter accumula-tions were affected by the K supply rates.Nevertheless, the root:shoot ratio increased(P,0.05)from0.20to 0.26following a decrease in the RAR from0.12to 0.03,respectively.Similarly,the total leaf area per plant was not affected(P.0.05).
Plants contained the same amount of N(P.0.05) irrespective of K supply rate(Table1).Plants that had adapted to the lowest K supply rate also contained the smallest(P,0.05)amount of K in shoot,root and nodules(Table1).As the dry matter yield was not affected by the K supply,this difference was caused by a lower K(P,0.05)concentration in plant dry matter at the lowest RAR.Plants with the lowest K supply rate compensated by increasing their Na uptake(Table1). Thus,the K supply rate had no effect on the combined K and Na content in roots(P?0.28)although the com-bined K and Na content was still reduced in nodules (P?0.04)and shoots(P?0.0001).Expressed on a molar basis,the Na substituted36%of the K in the shoots,100%in the roots and50%in the nodules when comparing the lowest and highest RAR. Irrespective of the K supply rate,plants maintained (P.0.05)the same N2-fixation per unit root FW and per unit nodule dry matter(Table2).This indifference was associated with the high EAC of70%and an Ar-ID in nitrogenase activity of23%.
The photosynthetic apparatus maintained or increased its capacity for CO2-fixation.Reducing the K supply rate from0.12to0.03caused an increase(P,0.05)in net photosynthesis per unit shoot area as well as per plant.
442Physiol.Plant.119,2003
The ratio of total net CO2-fixation to N2-fixation was much higher at a lower than at a higher K supply (Table2),suggesting a regulatory inhibition of N2-fixation following reduced N demands.
The shoot morphology changed with K supply in the sense that the SLA was reduced(P,0.05)at the highest RAR(Table2).Consequently,the ratio of total net CO2-fixation to N2-fixation was much higher at a lower than at a higher K supply rate(Table2),suggesting a regulatory inhibition of CO2-fixation following reduced N demands. However,under steady-state conditions,the photosyn-thetic apparatus remained intact,as the efficiency by which the incoming radiation was utilized(F PSII)was not affected by K supply rates(P.0.05)(data not shown).Also the non-photochemical quenching(i.e.radiating heat;qNP) and the photochemical quenching remained unaffected (data not shown).
Short-term dynamic responses to K withdrawal
Total withdrawal of the K supply for22days increased (P,0.05)shoot dry matter yield of single white clover plants(Fig.1A).The root:shoot ratio was not(P.0.05) affected by the K withdrawal within the experimental period with mean values decreasing from0.31at the onset of the K deprivation to0.20on day22.The relative growth rates of shoots and roots were similar for the K-starved and the control plants at0.074dayà1for shoots and0.053dayà1for roots.
Withdrawal of the K supply did not affect the N concentration(Fig.2A)in roots and nodules.The N concentration in shoots was similar(P.0.05)(Fig.2A) but the N accumulation at the final sampling(Fig.1B) was higher in the white clover plants experiencing the K withdrawal compared to the controls(P,0.05).
With the withdrawals of the K supply,the accumula-tion of K in the plant tissue ceased completely(Fig.1C) whereas the controls continued to accumulate K.This was reflected in a significant reduction in the K concen-tration in shoots,roots and nodules(Fig.2B).The Na concentration,however,strongly increased when the K supply was withdrawn(Fig.2C).At the final sampling, Na had substituted23%of K in shoots,50%of the K in roots,and24%of K in nodules when expressed on a molar basis.
At the final sampling,nodule N constituted36%and 6.3%of total root-N and total plant-N,respectively,in the K deprived plants compared to29%and5.4%in the controls.The corresponding values for nodule K were 31%and4.9%in the K deprived plants compared to 13%and1.7%in the controls.
Nodule dry mass continued to increase in the control plants as well as in the K-deprived plants(Fig.3A)but compared to controls the K deprived plants increased their nodule mass by77%at the final harvest.Surpris-ingly the SLA increased significantly from140to162 when reducing the K supply rate from0.12to0.03RAR (Table2),respectively,but when experiencing an abrupt withdrawal of K,the SLA of the white clover plants did not develop significantly differently from controls (Fig.3B).
Nitrogenase activity declined by24%per plant basis under continuous K supply,whereas plants that had their K supply withdrawn had a decline of55% (Fig.4A).However,as these K deprived plants increased their nodule mass compared to controls,their nitrogen-ase activity was reduced on a nodule mass basis (Fig.4B).On the last sampling occasion,nitrogenase activity per unit root as well as per unit nodule was
Table1.Nitrogen,potassium,and sodium accumulation(mg per plant)and content(%)in plant dry matter of white clover adapted to growth under different long-term steady-state K treatments controlled by the relative daily addition rate of K.Values are means of four observations (±SE).
Treatment
(g K gà1K dayà1)Shoot N
(mg)
Root N
(mg)
Nodule N
(mg)
Shoot K
(mg)
Root K
(mg)
Nodule K
(mg)
Shoot Na
(mg)
Root Na
(mg)
Nodule Na
(mg)
0.03111a19a8.4a34.7a 3.1a0.7a16.2a 5.4a0.5a
0.06112a17a9.5a65.9b 5.9b 1.7b 6.5b 3.4b0.3a
0.12106a16a7.2a185.2c8.0c 1.7b 1.7c0.3c0.1b
(%)(%)(%)(%)(%)(%)(%)(%)(%)
0.03 4.77a 4.08a 6.40a 1.52a0.68a0.49a0.69a 1.18a0.35a
0.06 4.39b 3.78b 6.38a 2.59b 1.32b 1.12b0.25b0.75b0.19b
0.12 4.05c 3.96a 6.11a 3.26c 1.96c 1.48c0.07c0.07c0.05c Different superscripts indicate differences on a5%level.
Table2.N2-fixation and net photosynthesis in white clover plants growing under different long-term steady-state potassium treatments controlled by the relative daily addition rate of K.Values are means of four observations(±SE).
Relative addition rate (g P g-1P day-1)N2-fixation
(m mol N2g-1
root FW h-1)
N2-fixation
(m mol N2g-1
nodule DW h-1)
Net photosynthesis
(m mol CO2m-2
leaf surface area s-1)
Net
photosynthesis
(m mol CO2s-1
plant-1)
Ratio net
photosynthesis:
N2-fixation(mmol
CO2m mol-1N2)
Specific leaf area
(cm g-1DM)
0.03 3.27(0.63)a160(24)a 6.58a1069a1301a162a
0.06 4.97(0.82)a196(61)a 5.84ab879ab786a151ab
0.12 3.26(0.31)a133(13)a 4.98b694b671a140b
Different superscripts indicate differences on a5%level.
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significantly reduced(P,0.045).The EAC was not affected by K supply(data not shown),maintaining mean values of69%.However,the Ar-ID of the H2efflux from the nodules increased by78%in plants experiencing a withdrawal of K compared to controls(Fig.5).
Net CO2-fixation per shoot of plants subjected to K starvation was higher than that of the controls (P,0.05),whereas the specific CO2exchange per unit shoot area was not affected by P supply(Fig.6).Specific CO2exchange decreased with plant age in both treat-ments throughout the22-day experimental period.The efficiency by which the incoming radiation was utilized (F PSII)was similar in K-deprived plants and controls (Fig.7).However,the non-photochemical quenching (i.e.radiating heat;qNP)was lower in the P-deprived plants(Fig.7),while the photochemical quenching remained unaffected(data not shown).
The ratio of total net CO2-fixation in shoots to total N2-fixation in roots was independent of K supply,sug-gesting a finely tuned interregulation of N2-fixation and photosynthetic CO2-fixation(Fig.8).
Discussion
Access to K may affect N2-fixation in grassland legumes in two ways:(1)K availability may become so low that legume growth will be affected;or(2)the competitive situation in grassland mixed swards will disadvantage the legume.In both cases,the consequence of
reduced
Fig.1.Dry matter yield(A)and content of nitrogen(B)and
potassium(C)in shoots(&,&)and roots(*,*)of white clover
plants growing with a continuous supply of potassium(&,*)or
abruptly deprived of potassium(&,*)following withdrawal of the
external potassium supply.Values are means of four observations.
Bars represent±SE
.
Fig.2.Concentration of nitrogen(A),potassium(B),and sodium
(C)in shoots(&,&),roots(*,*)and nodules(&,&)of white clover
plants grown with a continuous supply of potassium(&,*,&)or
abruptly deprived of potassium(&,*,z)following withdrawal of the
external potassium supply.Values are means of four observations.
Bars represent±SE.
444Physiol.Plant.119,2003
legume growth may affect the N input to agroecosys-tems,thus reducing the productivity of the systems as well as changing the fodder quality of the herbage.
The effect of K deficiency on N 2-fixation in white clover may manifest itself in short-and long-term responses.In the present study,these responses were investigated by applying two experimental approaches to imitate a steady-state K supply situation and a sudden change in K availability.Both approaches resulted in similar relative growth rates.Steady-state K supply experiment
In plants,whose dry matter accumulation was not yet affected by the relative daily K additions,root growth was favoured compared to shoot growth at low K supply but the N 2-fixation per unit root mass and per unit nodule mass was unaffected (Table 2).This is in accor-dance with the resource-ratio hypothesis where,under a steady-state situation in K availability,clover is expected to allocate resources preferentially to the roots (Bloom et al.1985,Chapin et al.1987).However,growth of the nodules was unaffected by the K supply rate.
A critical minimum value of 1.7%K in white clover shoots has been reported (Evans et al.1986).The 1.5%K in shoots from the 0.03RAR treatment (Table 1)together with the change in root:shoot ratio indicate that plants were limited by K deficiency.On the other
hand,RAR 0.12was not seen as leading to excess of K as concentrations of 3.5%K are often observed in white clover shoots under fertile field conditions (H?gh-Jensen,unpublished data).However,regulatory adjustments had affected the resource uptake rates as well as https://www.doczj.com/doc/fd18498008.html, photosynthesis per plant and per unit leaf area increased with reduction in the rate of K supply and the plants under low K supply rate increased their
concentration
Fig.3.Nodule mass (A)and specific leaf area (B)of white clover plants growing with a continuous supply of potassium (*)or abruptly deprived of potassium (*)following withdrawal of the external potassium supply.Values are means of four observations.Bars represent±SE
.
Fig.4.Nitrogenase activity per unit root fresh weight (A)and nitrogenase activity per unit nodule dry weight (B)in white clover plants growing with a continuous supply of potassium (*)or abruptly deprived of potassium (*)following withdrawal of the external potassium supply.Values are means of four observations.Bars represent±SE
.
Fig.5.Argon-induced decline in nitrogenase activity in white clover plants growing with a continuous supply of potassium (*)or abruptly deprived of potassium (*)following withdrawal of the external potassium supply.Bars represent±SE ;n ?4.
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445
of N in the shoots (Table 1).SLA increased when the rate of K supply was reduced (Table 2)despite a strong decline in the concentration of K in the shoots although plants experiencing a reduction in K uptake increased their Na uptake (Table 1).
The higher ratio of net CO 2to N 2-fixation,following adjustment to lower K supply rates,together with the maintenance of the N 2-fixation per unit root and nodule mass (Table 2),suggests that even mild K deficiency induced a feedback reduction of symbiotic N 2-fixation (Oti-Boateng and Silsbury 1993,Parsons et al.1993,Hartwig et al.1994).This inhibition may be caused by a reduced demand for N by the clover plants under
long-term steady-state conditions,as found by Neo and Layzell (1997).A similar conclusion was reached by H?gh-Jensen et al.(2002)who found high concentrations of asparagine in phloem extracts from P-deficient plants and by Almeida et al.(2000),who measured the asparagine concentration in P-deficient and control white clover plants grown with inorganic N supply.Short-term K deprivation experiment
As the clover plants in this experiment were older and therefore larger when subjected to K deficiency than the plants from the steady-state experiments,responses
to
https://www.doczj.com/doc/fd18498008.html, photosynthetic CO 2fixation per plant (*,*)and per unit leaf area (&,&)in white clover plants growing with a continuous supply of potassium (&,*)or abruptly deprived for potassium (&,*;)following withdrawal of the external potassium supply.Bars represent±SE ;n ?
4.
Fig.7.PSII quantum yield efficiency (&,&)and non-photochemical quenching coefficient (*,*)in leaves of white clover plants growing with a continuous supply of potassium (&,*)or abruptly deprived of potassium (&,*)following withdrawal of the external potassium supply.Mean SE is indicated;n ?12.
446
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K starvation were expected to occur with some delay.Initially,the plants had a high K status but after the withdrawal of K the K content was rapidly diluted in shoots and roots,in particular,but also in nodules (Fig.2B),which indicate that nodules do not constitute a preferential sink for K in white clover.Twelve days after K withdrawal,the concentration of K in roots and nodules was less than 1%.After another 5days,the concentration of K in shoots had also decreased to less than 1%.However,after the withdrawal of the K supply,the dilution of the combined content of K and Na took place at a much slower rate than K alone (Fig.2B,C).Na was not supplied to compensate completely for K but apparently it compensated sufficiently to ensure normal plant growth (Figs 1and 3)although the nitrogenase activity in the nodules (Fig.4)was https://www.doczj.com/doc/fd18498008.html,parison of short-term and long-term responses of white clover to K supply
Comparison of the results obtained in the present study using two complementary experimental approaches shows that,importantly,white clover plants were able to cope with K deprivation;dry matter yield as well as N concentration in dry matter were maintained (Table 1;Figs 1and 2A)in all tissue in agreement with other studies as reviewed by Duke and Collins (1985).Further,it is seen that responses of white clover to a lack of K supply depend on the duration of the stress as the effects displayed by plants subjected to steady-state low K supply exhibited a preferential allocation to roots,which were not observed in clover plants experiencing an abrupt termination of their K supply.However,the latter plants downregulated the specific nitrogenase activity of their nodules,i.e.per nodule mass,in contrast to those plants adapting to a steady-state low K supply.
Further,both experimental approaches showed that K and Na have complementary effects on plant growth,in agreement with Marschner (1971),and that Na substi-tuted 27–45%of the K in control plants when expressed on a molar basis.
The K concentrations in plant tissue at the end of the steady-state experiments were similar to the K concen-tration of the second series after approximately 12days of K-stress.Thus,the K-depletion developed further after abrupt withdrawal of K compared to a steady-state low supply of K.Consequently,the K concentration in the plant tissue of the second series was very low at the end of the experimental period:0.61,0.65,and 0.64%in roots,shoots and nodules,respectively.First,this is significantly below the critical minimum value of approximately 1.7%in shoots (Evans et al.1986).Second,the values are not significantly different,which is in accordance with the mobility of K.Third,the decrease in K concentration following the withdrawal of access to K in full growing white clover was dramatic,dropping from 4.7%in shoots at sufficient conditions to 2.0%8days later.This suggests a rapid depletion of the stored K in the vacuoles.
The unchanged EAC in white clover under K steady-state starvation suggest that K did not directly affect the diffusion barrier of the nodule (Sheehy et al.1983,Serraj et al.1995).However,regulation took place after the withdrawal of the K supply as the nitrogenase activity was reduced and the net CO 2-fixation was maintained causing an increased ratio of total net CO 2-fixation to N 2-fixation (Fig.8).However,possibly Na has compensated for K deficiency in the N 2-fixation process.The counteracting regulations of reduced N 2-fixation in response to K deprivation (Fig.4),the increase in nodule mass (Fig.3)and the increase in the Ar-ID in H 2efflux from the nodules (Fig.5)after abrupt withdrawal of K support this
possibility.
Fig.8.Ratio between total net CO 2fixation and total N 2fixation per white clover plant growing with a continuous supply of potassium (*)or abruptly deprived of potassium (*)following withdrawal of the external P supply.Bars represent±SE ;n ?4.
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447
Plants in both experimental set-ups exhibited an earlier physiological ageing when well supplied with K compared to K-deprived plants.This is reflected in a reduced SLA under steady-state well-supplied K conditions and smaller dry matter yields and N accumulations.This may be due to an interaction in nutrient access because the clover plants in all cases had access to Na and absorbed substantial amounts of Na when experiencing K deficiency(Table1, Fig.2B,C),possibly causing an additional effect of Na on growth(Mengel and Kirkby1980,Marschner1995).A change in the proportion of active to inactive nodule tissue is also possible(Fig.3A).
CO2-flux on shoots clearly decreased with age,but more in the K-sufficient plants that in the K-stressed.This decrease in CO2-flux may be caused by a combination of several processes.In the first series of experiments,no difference was observed in chlorophyll fluorescence of the shoots.In the second series,however,where K-deprivation developed more critically,the more rapid phenological ageing observed in the control plants may be dominant, as the photosynthetic efficiency seems to decrease in the control plants relative to the K-stressed plants.In addition, the non-photosynthetic quenching seems to be higher in the control plants,leading to the lower efficiency.This decrease in efficiency is,however,more a slow adjustment of the PSI and PSII to the actual light intensity indicating that the traps were slower to become oxidized again,i.e.to recover from a saturating pulse(Schreiber et al.1995).
In summary,the present study has shown that a low K status initiates whole plant adaptive responses in white clover.Changes in the relative growth of shoots,roots and nodules,rather than changes in the resource uptake rates per unit mass or area of these plant parts,appear to take place.Thus,the duration of K-deprivation and plant age may influence experimental results significantly.A not yet fully understood regulatory N-feedback mechanism apparently affects the regulation of nodule growth in K deficient white clover plants.As the K-deprived plants were not N-deficient,the observed reductions in shoot:root ratio were directly induced by a low K content in the plant tissue,rather than by a low N status.A strong comple-mentarity between K and Na uptake was found to partly alleviate the effect of K deprivations.Thus,future work on K deprivation should include Na as an experimental factor and definitions of threshold values of K concentrations should consider Na.
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Edited by J.I.Sprent
Physiol.Plant.119,2003449
(wuwei1970的博客) 原来心肌复极、静息状态的电活动分别是由不同类型的钾通道负责：高钾、低钾血症对心肌兴奋性的影响 问题1：高钾血症时，心肌复极时钾通道通透性增大，钾外流变快，但心肌静息状态时钾外流却减慢，两者不是矛盾吗？ 问题2：低钾血症时，为什么骨骼肌、平滑肌的兴奋性下降、而心肌细胞的兴奋性升高？ 答：可兴奋细胞（心肌、骨骼肌、平滑肌等）细胞膜上钾通道电流越强，钾外流越多，细胞内负电荷就越多，细胞的兴奋性越低;反之，钾外流越少，细胞内负电荷越少，细胞的兴奋性就越高。 心肌细胞的钾通道种类多，分为： 1. 电压依赖性钾通道，包括： Ik (延迟外向整流钾通道）：Ikr（快激活整流钾电流）, Iks（慢激活整流钾电流）, Ikur （ultra-rapid激活整流钾电流) Ik1(内向整流钾通道(inward rectifier potassium current），Ito (瞬时外向钾通道） 2. 配体/ 受体激活的钾通道，包括：IkATP (ATP依赖性钾通道）, IkAch（乙酰胆碱依赖性钾通道, IkAA（花生四烯酸依赖性钾通道） 上述各种钾通道，在心肌细胞的正常电生理活动和病理状态下的电活动中各自发挥其特定的作用。一般而言，电压依赖性钾通道和IkAch在心肌细胞的正常电生理活动中起重要作用，而在心肌缺血等病理条件下，配体/ 受体激活的钾通道如IkATP，IkAA等变得重要。 心肌细胞动作电位复极化及静息膜电位的形成，分别由不同类型的钾通道参与： （1）心肌细胞动作电位复极相的主要离子流取决于Ikr（快激活整流钾电流）, 其辐值大小决定了动作电位复极的速率。细胞外钾离子浓度变动对心肌Ikr的通透性会产生影响，即: 细胞外低钾时，心肌细胞Ikr变弱，钾外流减少，复极变慢，故心肌收缩性（平台期钙内流）
() 原来心肌复极、静息状态的电活动分别是由不同类型的钾通道负责：高钾、低钾血症对心肌兴奋性的影响 问题1：高钾血症时，心肌复极时钾通道通透性增大，钾外流变快，但心肌静息状态时钾外流却减慢，两者不是矛盾吗 问题2：低钾血症时，为什么骨骼肌、平滑肌的兴奋性下降、而心肌细胞的兴奋性升高 答：可兴奋细胞（心肌、骨骼肌、平滑肌等）细胞膜上钾通道电流越强，钾外流越多，细胞内负电荷就越多，细胞的兴奋性越低;反之，钾外流越少，细胞内负电荷越少，细胞的兴奋性就越高。 心肌细胞的钾通道种类多，分为： 1. 电压依赖性钾通道，包括： Ik (延迟外向整流钾通道）：Ikr（快激活整流钾电流）, Iks（慢激活整流钾电流）, Ikur（ultra-rapid 激活整流钾电流) Ik1(内向整流钾通道(inward rectifier potassium current），Ito (瞬时外向钾通道） 2. 配体/ 受体激活的钾通道，包括：IkATP (ATP依赖性钾通道）, IkAch（乙酰胆碱依赖性钾通道, IkAA（花生四烯酸依赖性钾通道）
上述各种钾通道，在心肌细胞的正常电生理活动和病理状态下的电活动中各自发挥其特定的 作用。一般而言，电压依赖性钾通道和IkAch在心肌细胞的正常电生理活动中起重要作用，而在心肌缺血等病理条件下，配体/ 受体激活的钾通道如IkATP，IkAA等变得重要。 心肌细胞动作电位复极化及静息膜电位的形成，分别由不同类型的钾通道参与： （1）心肌细胞动作电位复极相的主要离子流取决于Ikr（快激活整流钾电流）, 其辐值大小决定了动作电位复极的速率。细胞外钾离子浓度变动对心肌Ikr的通透性会产生影响，即: 细胞外低钾时，心肌细胞Ikr变弱，钾外流减少，复极变慢，故心肌收缩性（平台期钙内流）＼自律性（复极4相自发钠内流）增强; 反之，细胞外高钾时，Ikr变强，钾外流增多, 复极变快，心肌收缩性、自律性下降。 (2) 心肌细胞静息膜电位（正常：-90mv) 则主要受Ik1(内向整流钾通道)及背景钠内流的影响（参见人卫，朱大年等主编的“生理学”第8版P101-102）这一点与骨骼肌、平滑肌不同，骨骼肌、平滑肌无Ik1）。整流是引用物理学上的名词,指电流易向一个方向流动,而不易向反方向流动；内向整流（inward rectification)是指正离子易从细胞外流入胞内,而不易从细胞膜内流向膜外（参见人卫，姚泰的“生理学”第5版P88）。Ik1(内向整流钾通道)开放程度受膜电位的影响。低钾血症，心肌细胞膜发生超极化趋势时（静息膜电位在-85mv 以下），Ik1激活，促进钾内流的电场力大于促进钾外流的浓度势能，钾则内流；以阻止钾外流，而基础的钠内流又使膜部分去极化，从而使心肌细胞的静息电位绝对值减小，故兴奋性增高。而骨骼肌、平滑肌无Ik1,所以低钾血症骨骼肌、平滑肌发生超极化趋势时，无法阻断钾的外 流，从而最终引起超极化现象，骨骼肌、平滑肌的兴奋性下降。 (3) 高钾血症,心肌细胞静息状态时根本无超极化发生（因为高血钾导致心肌细胞静息状态时细胞内外钾浓度差缩小，钾外流变少，静息膜电位绝对值变小，如为-75mv),心肌兴奋性会因为较接近阈电位而暂时升高，但当静息膜电位过于接近阈电位-60mv时，快钠通道完全失活，心肌兴奋性下降。 综合（1）（3）点可回答问题1，第（2）点可回答问题2。 总之，细胞外钾变化对心肌的影响是：低钾血症时，钾外流变少，心肌兴奋性、自律性升高; 高钾血症时，（复极）钾外流变多，自律性下降，心肌兴奋性先升后降。因自律性就是心肌自律细胞的兴奋性，故简而言之，低钾血症时心肌兴奋性升高，高钾血症时心肌兴奋性降低！
协同参与调节其复极过程, 并以延迟整流钾通道（delayed rectifier potassium cur- rent,IK）的作用最为重要。20 世纪60 年代末,Noble 和Tsien 首次在羊浦 成分, 分别称为IK1和IK2［1］。1990 年Sanguinetti等观察了E24031对豚鼠单 不同的敏感性分别命名为快速激活的延迟整流钾通道（rapidly activated delaye ctivated delayed rectifier potassuim curr- ent, IKs）。目前，普遍认为IK 包含3个成分，除了上述的两种通道以外，还包含超快激活的延迟整流钾电流（ul
至少两倍。有研究证明在KCNQ1/KCNE1通道上有两个功能各不相同的PKC作用位点，KCNE1上的磷酸化位点N102是PKC对IKs进行调控的作用位点［13］。除此之外，PKC预调控会阻碍PKA对IKs的调控，而且PKC对于已经因为PKA调控而增强的IK s电流不产生作用，这说明PKA和PKC对IKs的调控是互相排斥的。 4 IKs阻断剂 IKs对甲磺酸盐类有抵抗作用，但可被色原烷醇类（293B、HMR-1556）,吲 哚帕胺，硫喷妥钠，异丙酚和地西泮（L-768、673，L735、821或L-7）选择性阻断［15～17］。此外，色原烷醇类对于开放通道的阻断作用具有对映选择性，（-）3 R，4S-293B和（-）3R，4S-HMR1556为有效的IKs选择性阻断剂就是很好的例子［1 8］。 IKs一开始就是Ⅲ类抗心律失常药的非常有吸引力的作用靶点。这是因为I Ks由于它的缓慢失活特性在高速驱动频率下会出现累积，于是IKs阻断剂有希望在高频率下更有效地延长APD。实际上，在人心室肌细胞中，293B延长APD和不应期且与频率无关，所以认为IKs阻断剂与IKr阻断剂相比较而言，致心律失常性更低［19］。另外，因为IKs激活发生0mV附近，而这个电压较浦肯野纤维的动作电位更正，IKs在这个水平上应该不会延长APD。而在心室肌细胞中，平台电位更正（≈+20mV），使得IKs本质上更多地被激活，反而阻断IKs可能会明显延长APD。这两种作用的净结果可能是减少了极化过程中药物诱发的离散程度并降低致心律失常性。 IKs阻断剂延长心脏APD和QT间期，并能缓解已治愈心肌梗死动物由于急 性冠脉缺血、运动而引发的室性快速心律失常，这种QT间期延长可因为受到β-肾上腺素的刺激而得到增强。犬左心室动脉灌流中，293B延长心室APD和QT间期， 并且并不引发TdP，然而在293B存在的情况下，异丙肾上腺素会缩短心内膜和心外膜上的ADP，但对M细胞没有影响，这样就加强了复极化过程中的透壁不均一性， 引发TdP［20］。这就是为什么β-肾上腺素对LQT1及LQT5型病人有治疗作用，但