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									Plant Physiol. (1979) 64, 660-662
0032-0889/79/64/0660/03/$00.50/0


Short Communication

Effect of Shoot Removal and Malate on the Activity of Nitrate
Reductase Assayed in Vivo in Barley Roots (Hordeum vulgare cv.
Midas)
                                                                        Received for publication February 5, 1979 and in revised form May 11, 1979

             CELIA E. DEANE-DRUMMOND"' AND DAVID T. CLARKSON
             Letcombe Laboratory, Wantage, Oxon, United Kingdom
             CHRISTOPHER B. JOHNSON2
             School of Agriculture, University of Nottingham, Sutton Bonington, Leics, United Kingdom

                             ABSTRACT                                         10 mm N03- was given 6 days after germination and all enzyme
                                                                              assays were made 3 days later. Pretreatment solutions containing
   There is a diurnal variation of nitrate reductase activity (NRA) measured 10                                    nutrient solution were adjusted
in vivo in barley roots (Hordum vdgare cv. Midas). In intact plants to mm organicpH 5.4 with strength before use. Amino acids were
                                                                                              acids in full
                                                                                 pH 5.8 to                  NaOH
receiving a 16-hour photoperiod, NRA increases when the light     is switched
                                                                              measured using a ninhydrin method (17).
on, reaches a maximum value after 7 to 8 hours, and thereafter declines.
Sboot removal (detopping) at the start of the pbotoperiod prevents the rise                               ENZYME ASSAYS
in NRA; detopping after 5 hours lght leads to a rapid fall in NRA. The
inclusion of 10 mll r maiate in the exteral medim causes a rise in               In Vivo Assay. Tests in our laboratory showed that the following
NRA in plats detopped at the              ning of the photoperiod and thus    conditions were optimal. Incubation medium contained 50 mm
seems to subsdtute partially for the illuminated shoot. Oxalate, fumarate, phosphate buffer, 1% propanol (v/v), 100 mm nitrate at pH 8.0.
and tartrate did not have this effect. Preincubation of the roots of intact Roots were transferred to glass bottles containing cold incubation
plants with 10 milimolar malate for 3 hours, prior to detopping, causes an medium (4 ml/100 mg root) and vacuum-infiltrated for 4 min.
increase in the flux of amino acids into the xylem sap of detopped roots. Incubation was for 20 min at 27 C in the dark and the reaction
                                                                              terminated by boiling for 10 min. Suitable blanks and zero time
                                                                              controls were run for each assay. After cooling, nitrite was meas-
                                                                              ured in a 1-ml aliquot by adding I ml sulfanilic acid (1% in 3 N
                                                                              HCI) and then I ml N-l-naphthylethylene diamine dihydrochlo-
                                                                              ride (0.05%). The optical density at 540 nm was measured at least
                                                                              2 h later.
   Diurnal rhythms in NRA3 in leaves (1, 15), as well as apparently              In Vitro Assay. The extraction medium contained 3% casein, 10
endogenous rhythms in activity (16) are well known. A recent mM cysteine, I mm EDTA in 50 mm phosphate buffer at pH 7.5.
publication (14) has established that the removal of shoots from The ratio of tissue to extractant was 1:5 (w/v); the extract was
cotton roots led to a marked decline in NRA assayed (in vivo) and made at 3 C using a pestle and mortar and then centrifuged for 10
in sugar content in the roots, but it is not clear if any indirect min at 18,000g. Aliquots of the supernatant (0.1 ml) were mixed
light-mediated control of NRA is sustained at different times with a solution containing NADH (0.2 mg/ml), 10 mm KNO3
during the photoperiod. This is of particular importance for made up in 100 mm phosphate buffer at pH 7.8 giving a final
interpretation of relatively long term experiments which involve volume of I ml. The mixture was incubated for 15 min at 27 C
analysis of amino acid fluxes into the xylem sap of detopped roots and was then treated with 0.5 ml of boiling 0.3 M zinc sulfate.
(13). The main aim of this work was to investigate the extent to After cooling, 0.2 ml of I N NaOH was added and the mixture
which the maintenance of NRA in the roots of barley seedlings is centrifuged for 10 min. Nitrite in the supernatant was determined
dependent on shoots at different times during a 16-h day regime. as in the in vivo assay.
Further experiments have made it possible to draw tentative
conclusions about a role of malate in this root-shoot interaction.                             RESULTS AND DISCUSSION
                   MATERIALS AND METHODS                                         The results presented here have to be interpreted in the light of
                                                                              the limitations and advantages of the assay technique used (3).
   Methods for growing barley plants Hordeum vulgare (cv. Midas) The in vivo assay, unlike the in vitro assay, does not have an
and collecting xylem sap from detopped plants have been de- exogenous supply of reducing power (3). Moreover casein and
scribed elsewhere (4). Full strength nutrient solution containing cysteine protectants added to the in vitro assay prevent inactivation
   ' C. E. Deane-Drummond thanks the Science Research Council for the of enzyme during the assay period,
                                                                                                                      whereas in the in vivo assay
award of a C.A.S.E. Studentship.                                               the propan-l-ol treatment could release endogenous inhibitory
   2Prewnt address: Botany Department, Plant Science Laboratories, substances. a marked peak in NRA assayed in vivo was found
                                                                                  In roots
Reading University, Reading, England.
   '
     Abbreviation: NRA: nitrate reductase activity.                            during the first 12 h of light (Fig. 1). The increase in enzyme
                                                                       660
Plant Physiol. Vol. 64, 1979                   N03- REDUCTION IN BARLEY ROOTS                                                                                       661
                                                                            the Krebs cycle, the fact that fumarate did not increase NRA
                                                                            assayed in vivo implies that exogenous malate sustains NRA
                                                                            activity via the cytoplasmic rather than the mitochondrial pool.
       I--                                                                     Although there are many steps between the reduction of nitrate
                                                                            and its export as amino N in the xylem sap, the over-all capacity
       ..
                                                                            for providing reduced nitrogen to the shoots can be measured by
       I--                                                                  analysis of amino acids in xylem sap (13). Pretreatment for 3 h
        .0
                                                                            with malate also increased the rate of export of amino acids into
         E                                                                  xylem sap (Table III) indicating that there is an effect of malate
                                                                            on nitrate assimilation: there was no corresponding effects of
                                                                            fumarate and tartrate pretreatments (unpublished results). The
                                                                            results are compatible with malate having its main effect on nitrate
                                                                            assimilation in the intact plant, but the possibility cannot be ruled
                           TIME hours after light on
                                                                            out that it may also affect subsequent steps in amino acid transport.
                                                                            Malate pretreatment was most effective when roots were excised
   FIG. 1. Effect of light, shoot removal, and exogenous malate (10 mM) early in the photoperiod; this would be expected if malate shortage
on nitrate reductase activity assayed in vivo in barley roots. In all cases
                                                                            was most significant during the first 7 h of light. Further experi-
roots were kept in darkness throughout the experiment. All values mean
of at least four replicates, standard errors < 10%o mean. Arrow indicates ments
                                                                                    are needed to establish this point.
shoot removal.                                                                 In vitro measurements showed no significant decline in NRA
 activity appeared to be related to transfer of plants to the light         Table I. Comparison of NRA in Vitro in Barley Rootsfrom Intact and
 rather than to any circadian rhythm, since there was no increase                                      Detopped Plants
 in plants kept in continuous light between 24 and 29 h. Where the           The 18,000g pellet was discarded and all assays were carried out using
 shoots had been removed before starting the light treatment, roots       supernatant. Enzyme was extracted at 0 to 3 C and the reaction started by
 did not show this peak unless malate was included in the external        adding enzyme preparation to assay mixture at 27 C.
 medium. The immediate decline in NRA after 5 h light which
                                                                              Time After                                         NRA
 resulted from detopping was partially prevented if malate was                 Light On                 Intact plants                        Detopped plants?
 included in the external medium at the time of detopping and                      h                               gmol ar*ue/h-gfresh weigh
 almost completely prevented if a 3-h malate pretreatment was
 given. After 7 h there was a fall in NRA; factors other than                      7               1.74 ± 0.03                                1.73 ± 0.08
 limiting malate supply were likely to be involved since this fall in              9               1.73 ± 0.05                                1.71 ± 0.05
 NRA was also found in malate-pretreated roots. Diurnal changes                   11               1.93 ± 0.04                                2.2 ± 0.04
 in inhibitory enzymes have been observed in rice roots (18) and              Plants detopped 7 h after light on.
 similar changes may possibly have complicated our experiments.             b Mean of four replicate determinations ± S.E.
 No evidence was found of any loss of NRA in vitro between 7 and
 11 h (Table I); although under the assay conditions used in vitro         Table II. Effect of Various Organic Acids (10 mM) on NRA in Vivo in
 the nitrate reductase inhibitor activity was probably undetected.                         Barley Roots Following Shoot Removal
    Removal of shoots or keeping the plants in darkness had similar         Organic acids, adjusted to pH 5.5, were added to full strength nutrient
effects on root NRA assayed in vivo, implying that the shoot             solution 3 h before detopping.
 regulates the activity of the root enzyme directly or indirectly. The
role of light in nitrate reduction in plant shoots has been discussed    Hours after light on                 8                    9
in detail in a comprehensive review (1), but without mention of          Hours after detopping                1                    2
any possible effect in the roots.                                        No additive                    0.37 ± 0.04a          0.20 ± 0.04
   Even though malate has an effect on NRA assayed in vivo, its          + Fumarate                     0.20 ± 0.05           0.17 ± 0.05
role, if any, in the intact cell remains obscure. A decrease in          + Oxalate                      0.23 ± 0.07           0.07 ± 0.02
malate content has been found in roots after excision (2) and            + L-Malate                     0.65 ± 0.02           0.33 ± 0.07
reports by other workers have shown not only that there is a             + DL-Tartrate                  0.34 ± 0.05           0.19 ± 0.04
gradient of malate between roots and shoots (with more malate in         Intact plants                      0.65b                0.45b
shoots) (9), but also that malate can act as a source of reducing           a
                                                                              Nitrate production (umol/h.g fresh weight). Mean of four determi-
power for nitrate reductase in leaves (11, 12). Although work on         nations ± SE.
excised pea roots (5) suggests that malate does not provide                 b Mean of three experiments.
NADPH via malic enzyme during the induction of nitrate reduc-
tase it may possibly provide a source of NADH via cytoplasmic            Table III.    Effect of Malate Pretreatments on Amino Acid Flux into Xylem
malate dehydrogenase. The proposition that changes in levels of                                Sap of Detopped Barley Roots
malate in the root are too small to have a role in nitrate uptake           All values are from pooled samples from at least 10 9-day-old plants.
and reduction (7) neglects the fact that malate can exist as a                                                          Time of Excision After Light On (Hours)
divalent anion at pH values about its pKa and there could be a                                Sampling time
continuous flow of malate from the shoots. Malate could also                 Pretreatment     Hours aer ex-                                             7

                                                                                                  cision
influence nitrate assimilation in the intact plant by changing the                                               Amino           Amino
                                                                                                                                acid flux'
                                                                                                                                              Amino          Amino
                                                                                                                 acids'                       acids'        acid flux'
availability of nitrate to the enzyme; e.g. some might exchange for                                               mM                            mM
nitrate in the vacuolar pool, or increase the uptake of nitrate into
the cells (2). This mechanism for malate action is unlikely during       No malate                 2               6.9           0.182          4.3          0.17
the in vivo assay since shortage of nitrate in the cells would be        Pretreatment              4               5.2           0.163          4.2          0.11
prevented both by the high external concentrations of nitrate (100       + L-Malate (10            2              12.6           0.32           6.1          0.25
mM) as well as propan-l-ol used.                                           mM)
   Organic acids other than malate depressed or had no effect on         Pretreatment for        4         12.2      0.41                       4.5          0.165
NRA of excised roots in vivo (Table II). That malate exists in             3h
separate cytoplasmic and mitochondrial pools is well established           a Glycine equivalent.
(10) and since fumarate could produce malate in mitochondria via           bFlux expressed as Fmol/h * g fresh weight root.
662                                             DEANE-DRUMMOND, CLARKSON, AND JOHNSON                                                               Plant Physiol. Vol. 64, 1979
following shoot removal even after 4 h (Table I). Presumably in                             2. BEN ZIONI A 1971 Nitrate uptake by roots as regulated by nitrate reduction products of the
                                                                                                 shoots. Physiol Plant 24: 288-290
our relatively short term experiments, changes in substrate pools                           3. BRUNETTI N, RH HAGEMAN 1976 Comparison of in vivo and in vitro assays of nitrate reductase
(e.g. nitrate or NADH) following shoot removal were more sig-                                    in wheat (Triticum aestivum) seedlings. Plant Physiol 58: 583-587
nificant than changes in enzyme protein levels, i.e. the capacity                           4. CLARKSON DT, MGT SHONE, AV WOOD 1974 The effect of pretreatment temperature on the
for nitrate reduction by the roots was altered, whereas the potential                            exudation of xylem sap by detached barley root systems. Planta 121: 81-92
                                                                                            5. FOWLER M, GS SARKISSIAN 1975 Malic enzyme as a source of NADPH for nitrate assimilation
remained unchanged. This contrasts with other reports (6), where                                 in roots. Plant Sci Lett 4: 41-46
after 48 h decapitated plants had less than 30%o activity of NR in                          6. FRITH Gi 1974 Light stimulated activity of nitrate reductase in apple roots. Plant Cell Physiol
vitro compared with intact plants. Other workers (e.g. 8) have                                    15: 153-155
found changes in ion uptake in roots following excision; K+ uptake                          7. FROST WB. DG BLEVINS, NM BARNETr 1978 Cation pretreatment effects on nitrate uptake,
declines and later recovers to rates found in intact plants, but in                              xylem exudate and malate levels in wheat seedlings. Plant Physiol 61: 323-326
                                                                                            8. GLASS DM 1978 Influence of excision and aging on K' influx into barley roots. Recovery or
our experiments there was small evidence of any corresponding                                    enhancement? Plant Physiol 61: 481-483
delayed recovery of NRA assayed in vivo. A decrease in sugar                                9. KIRKaY EA, AH KNIGHT 1977 Influence of level of nitrate nutrition on ion uptake and
concentration in roots following detopping may be a prime cause                                  assimilation, organic acid accumulation, cation anion balance in whole tomato plants. Plant
                                                                                                 Physiol 60: 349-353
of loss of NRA in vitro (6) and in NRA assayed in vivo in cotton                           10. LiPs SH, L BEEVERS 1966 Compartmentation of organic acids in corn roots. 1. Differential
roots (14). However, in barley roots there seems to be no shortage                               labeling of two malate pools. Plant Physiol 41: 709-712
of respiratory sugars for at least 6 h after excision; 02 uptake does                      11. MANN AF, DP HucKLESBY, EJ HEWITT 1978 Sources of reducing power for nitrate reduction
not slow significantly during this time (4). Further measurements                                in spinach leaves. Planta 140: 261-263
                                                                                           12. NEYRA CA, RH HAGEMAN 1978 Pathway for nitrate assimilation in corn (Zea mays L.) leaves.
of sugar and malate levels may clarify this point.                                               Plant Physiol 62: 618-621
   Our main conclusion is that the results of experiments with                             13. PATE IS 1973 Uptake, assimilation and export of nitrogen by the root. i Soil Sci Biochem 5:
excised roots should be interpreted with caution because of the                                   109-119
rapid loss of NRA assayed in vivo when the shoots are removed                              14. RADIN JW, LL PARKER, CR SELL 1978 Partitioning of sugar between growth and nitrate
and the marked diurnal periodicity in NRA in roots. The loss was                                 reduction in cotton roots. Plant Physiol 62: 550-553
                                                                                           15. SHIBATA M. M KOBAYASHI, E TAKAHASHI 1969 The possibility of photo-induced induction of
largely alleviated by supplying the plants with exogenous malate;                                 nitrate reductase in rice seedlings. Plant Cell Physiol 10: 337-348
but the mechanism of this effect is not clear.                                             16. STEER BT 1976 Rhythmic nitrate reductase activity in leaves of Capsicum annuum L. and the
                                                                                                  influence of kinetin. Plant Physiol 57: 928-932
                                 LITERATURE CITED                                          17. WYLIE EB, MJ JOHNSON 1962 Effect of penicillin on the ceil wall of E. coli. Biochim Biophys
                                                                                                  Acta 59: 450457
 1. BEEVERS L, RH HAGEMAN 1972 The role of light in nitrate metabolism in higher plants.   18. YAMAYA T, K OHIRA 1978 Nitrate reductase in activating factor from rice seedlings. Plant Cell
      Photophysiology 7: 85-113                                                                   Physiol 19: 211-220

								
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