Plant Cell Reports

Plant Cell Reports (1986) 5:368-371

©

Springer-Verlag 1986

Nitrate reductase activity and nitrate accumulation in in vitro produced axillary shoots, plantlets and seedlings of h'nus pinaster M . Faye, A. David, and A. L a m a n t Laboratoire de Biologie et Physiologie Vdg6tales, Universit6 de Bordeaux I, All~e des Facult6s, F-33405 Talence C6dex, France Received May 13, 1986 / Revised version received July 7, 1986 - Communicated by A. M. Boudet

Abstract The initial and induced in vivo Nitrate Reductase Activity, the nitrate accumulation by in vitro-produced axillary shoots and plantlets of Pinus pinaster were compared respectively with those of shoots collected from seedlings and whole plants. The usefulness of the nitrate of the medium used for in vitro axillary shoot formation is demonstrated by the oceurence of initial NR activity in the explants. When fed in a non in vitro situation with a 50 ram KNO_ solution, they have the same induced capacity to reduce nitrate as do shoots from seedlings, even though the latter accumulate less nitrate. Plants regenerated in vitro exhibit an ability to reduce nitrate similar to that of seedlings. In both types of plants, the Nitrate Reductase potential is greater in roots than in shoots. Abbreviation

:

NR = Nitrate Reductase BA : 6-Benzyladenine

Introduction Within the past ten years, research on micropropagation of Angiosperms (Brown and Sommer 1982) and Gymnosperms (David 1982) has been extensively developed. However, little attention was devoted to the mineral nutrition of cultures. Concerning nitrogen assimilation, Gymnosperms are thought to reduce nitrate predominantly in their roots and to translocate very little to the shoots (Martin et al. 1981; Smirnoff et al. 1984). One w o n d e ~ w h e ther nitrate is assimilated (a) in shoots cultivated in vitro on media commonly containing that ion as the main form of nitrogen supply, and (b) in plantlets originating from shoots produced in vitro. Since Nitrate Reductase is an inducible enzyme, a significant level of activity can be used as a good indicator of the uptake of nitrate by the organs and its availability in the cells. Therefore, NR activity has been assayed and nitrate content measured to determine whether the process of reduction is induced in axillary shoots by the presence of nitrate in the culture medium and to compare the capacity of newly-formed plants and seedlings to achieve the first rate-limiting step for nitrate assimilation. Nitrate is also known to act as an osmoticum (Smirnoff 1985). However, in the present paper, we have not considered this aspect. Materials and Methods Plant material. In vitro axillary shoots: Shoots (3 to 4 cm high)were obtained by axi!lary budding of primary explants(David

Offprint requests to: M. Faye

et ai.1978). These shoots have been multiplied for 5 years according to this method. In the basal medium, nitrogen was supplied as a mixture of nitrate (24.6 mM) and ammonium (6 mM). For NR activity assay shoots where sampled within a 30-day period following a subculture. Plantlets: Rooting of axillary shoots was performed as previously described (Rancillac et al. 1982) with a medium containing 3.3 mM nitrate plus 2 mM glutamine and 0.03 mM glycine as the nitrogen supply. After rooting, the plantlets were transferred (when the roots were 0.5 to I cm long) to fog-boxes and fed with distilled water in order to speed up root elongation. Their aerial parts were covered with a plastic hood which was removed for a longer period every 2 or 3 days through a total period of 2 weeks. The plantlets were harvested and analysed 2 to 3 weeks after the end of their acclimatization. Seedlings: Seedlings were used as control for in vivo NR activity assay. After sterilization of their surface with hydrogen peroxyde (30%) for 10 minutes, the seeds were germinated on filter paper, moistened with distilled water. Then, the seedlings with almost fully developed cotyledons were transplanted to fog-boxes and fed with distilled water for 4 to 5 weeks. Growth was achieved in a room programmed for 25/23°C day/ night temperature with a 16 h "day" period (Sylvania F40T 12 Gro-lux, 11.6 Wm -2) and an 8 h ~night" period. The seedlings were sampled when their shoots were 3 cm in height. In vivo NR activity assay Because of our unsuccessful attempts to determine in situ NR activity without exogenous nitrate,we decided to use an in vivo assay. The initial in vivo NR activity reveals the response to various conditions of culture. The induced NR activity measures the ability of the organs to reduce nitrate after NO 3- feeding according to the following procedure. Single shoots and plants (plantlets or seedlings)were fed with an aerated 25 or 50 mM KNO 3 solution, supplemented with chloramphenicol (50 mg.l-1). The inductive treatment lasted 16 hours including 7 hours of illumination (11.6 Wm -2) at 27°C. Plant material was always collected 6 hours after the beginning of the light period and sampled on ice (no longer than 5 min.). Each sample consisted of either four apical root segments (4 em in length, around 50 mg total fresh weight) or two shoots (approximately 400 mg total fresh weight). The standard incubation medium used (5 ml for roots and 3 ml for shoots) contained 100 mM KNO 3 in 100 mM K-phosphate buffer (pH 7.5) and either I% 1-propanol

369 for root assay or 0.015% triton X-IOO for shoot assay (Lawrence and Herrick 1982). Chloramphenicol (30 ug. ml -I) was routinely added to the basal medium (Aslam 1981). All the following steps were performed in the dark. Shoot samples in the vials were vacuum infiltrated for 8 min. Vacuum infiltration was not carried out for root assay since it did not increase NR activity. Anaerobiosis was achieved by flushing the vials with N 2 (output pressure: 150 K Pa) via hypodermic needles (Robin et al. 1983) for 2 min. The assay vials were incubated at 35°C in a shaking water bath for 2 hours. Following incubation, flasks were transferred to boiling water for 10 min. The amount of nitrite formed by the sample and released into a 2 ml aliquot of the solution was determined colorimetrically with I ml I% (w/v) sulfanilamide and I ml 0.02% (w/v) N-(1-naphtyl) ethylenediamine dihydrochloride. Absorbance was read at 540 rim. NR activity was expressed as ~mol NO 2- produced in a 2-hour period per gram of dry weight because of the large difference between dry matter percentages of in vitro axillary shoots and shoots from seedlings or plantlets. Each value represents the mean of 3 successive experiments with 5 replicates. Nitrate content The initial nitrate content is the quantity which accumulates in plant material in various conditions of culture. The induced nitrate content is the quantity in NO_--fed organs. J Dry material was extracted with 0 . 1 N NCI supplemented with 0.1% (w/v) HgCI 2 for 48 hours. Nitrate was analysed by an automated procedure of cadmium reduction (Treguer and Lecorre 1975) of nitrate to nitrite determined colorimetrically as described above. Results were expressed as ~mol NO B - per gram of dry weight. Each value represents the-mean of 3 successive experiments with 2 to 3 replicates. Results In vitro axillary shoots. A low initial NR activity (table I) was detected in axillary shoots cultivated on a medium containing

24.6 mM NO^--N. Nevertheless, this level of activity J was signiflcantly higher than that measured in shoots c~llected from seedlings growing on distilled water (control) [0.61 ~mol and 0.26 umol NO 2- (2 h g d. wt.) -I When the axillary shoots produced in vitro were fed for 16 hours with a 50 mM KNO 3 solution, they gained the same level of activity as the shoots collected from seedlings, submitted to identical inductive conditions [around 2.5 umol NO 2- (2 h g d.wt.) -I ]. When a nitrate solution at the same concentration as that of the culture medium was used for the inductive treatment of the axillary shoots, they exhibited a level of NR activity twice as much as that of the shoots cultivated for the same time on an agarified medium containing the same concentration of NO3--N. The initial nitrate content of both, shoots cultivated in vitro and shoots from seedlings fed with distilled water was similar and very low (table I). After an inductive treatment (16h, 50 mM KNO 3) axillary shoots accumulated 4 times as much nitrate as the control [78.3 and 20.1 ~mol (g d.wt.) -I, respectivelyJ--. On submitting axillary shoots to an inductive treatment with a 25 mM KNO 3 solution, the nitrate accumulation was found to be 18.7 ~mol g-1 d.wt., compared to the value for shoots cultivated on an agarified medium (0.55 ~mol g-1 d. wt.).

respectively].

Seedlings and plantlets In the roots of seedlings (control) the level of initial NR activity (table 2) was low [0.56 ~mol NO2-

(2 h g d . w t . ) - I ] although s l i g h t l y higher than t h a t in the shoots [0.16 ~mol NO2- (2 h d d . w t . ) - I ] . These organs did not accumulate nitrate. After an inductive treatment, the ability to reduce nitrate was high in both parts of the plants ~ o o t : 3.68; shoot : 1.16 ~mol NO 2- (2 h g d.wt.) - I ~ . Concomitantly, we noted a much greater nitrate content in the roots than in the shoots (244.8 and 10.4 ~mol g-1 d.wt. respectively). Initial NR activities, higher in roots than in shoots of plantlets (table 2) had the same level as those in the corresponding organs of the seedlings. Submitted to an inductive treatment the roots and shoots of the plantlets gained an equivalent capacity

Table I. In vivo NR activity and nitrate content of shoots. (I) Shoots collected from 8-week-old seedlings, grown on distilled water. (2) Medium for shoot culture: NO3--N (24.6 mM) + NH4+-N (6 mM). (3) Measurements at collecting time. (4) Measurements 16 h after subculture. (5) Measurements after supply of a 25 mM KNO 3 solution for 16 h. (6) Measurements after supply of a 50 mM KNO 3 solution for 16 h. Data are based on 3 successive experiments with 5 replicates, s = standard deviation and vc : variation coefficient. Different superscript letters on the same line indicate significant differences at the 0.05 level.

Parameter

Shoots from seedlings Initial (3)

NR activity ~mol NO 2(2 h g d.wt.)NO 3Bmol g-1 d.wt.

x s ve

(I)

In vitro Axillary shoots (2)

Induced 50 mM (6

Initial (4)

0.26 0.14 0.53

2.40 c 0.90 0.37

0.61 b O.17 0.28

1.13 0.08 0.07

2.53 0.50 0.19

0.33 O.11 0.33

20.10 b 6.20 0.31

0.55 a 0.30 0.54

18.70 3.20 0.17

78.30 19.00 0.24

25 mM (5)

Induced 5O mM (6)

370 Table 2. Seedlings and Plantlets in vivo NR activity and nitrate content. (I) 8-week-old seedlings grown on distilled water. (2) Plantlets obtained in vitro after rooting of axillary shoots. (3) Seedlings or plantlets fed for 16 h with distilled water. (4) Seedlings or plantlets fed for 16 h with a 50 mM KNO 3 solution. Data are based on 3 successive experiments with 5 replicates, s : standard deviation and vc = variation coefficient. Different superscript letters on the same line indicate significant differences at the 0.05 level. * Not detectable.

Roots Parameter

Seedlings (I)

NR activity

a

0.56

Initial

Shoots Plantlets (2) 0.98

a

Seedlings (I) b

O.16

Plantlets (2) 0.08

c

(3) ~mol

NO2

(2 h g d.wt.)

-1 Induced

s

0.04

0.37

0.02

0.03

vc

0.07

0.37

0.12

O.41

3.68 a

3.90 a

1.16 b

1.36 b

0.80

0.91

0.17

0.55

(4) 0.22 NO 3-

Initial

ND

0.23 *

3.99

0.15 a

ND

0.40 *

0.37

b

(3) pmol g

-I

1.30

d. wt.

0.24

0.32 Induced

244.80 a

209.70 a

0.65 10.40 b

62.70 c

(4) 32.20

15.90

5.74

38.60

0.15

0.08

0.55

O.61

to reduce nitrate as the corresponding parts of seedlings in the same conditions ~ 3 . 9 and 1.36 pmol NO 2(2 h g d . w t . ) - 1 , r e s p e c t i v e l y ~ . Whereas roots of plantlets and seedlings had the same nitrate content, shoots of plantlets accumulated more (62.7 and 10.4 umol g-1 d.wt., respectively). Discussion In vitro axillary shoots Since an initial activity existed in the shoots cultivated in the presence of nitrate, this ion was taken up from the medium in a sufficient quantity for triggering the process of reduction. Feeding the shoots for 16 hours with a 25 mM KNO 3 solution doubled the NR activity and induced a nitrate accumulation whereas with the same period on an agarified medium at the same concentration did not. These observations suggest that nitrate uptake from a gelled substrate is less than from a solution. Adding a gelling agent to a nutritive solution decreases matric potential, hence limits ion uptake which is closely linked with water influx in shoots cultivated in vitro. It has been demonstrated that a low matric potential diminishes the availability of any components of the medium (Debergh 1983) and the accumulation of BA in the explsnts of Picea abies (Bornman and Vogelmann (1984). Submitted to an inductive treatment (16 h, 50 mM KNO 3 ), the shoots originating from in vitro culture accumulate approximately 4 times as much nitrate as those collected from seedlings grown in an aerated atmosphere, in spite of the same rate of NR activity. In vitro formed leaves have been reported to have less cuticule, less epicuticular waxes and an impaired stomatal functioning (Wetzstein and Sommer 1982; Conner

and Conner 1984). This could result in an increased hydric flux leading to a greater nitrate accumulation. On the other hand more osmoticum could be required in the cells of shoots grown in vitro. Indeed, histological observations showed that Pinus pinaster shoots grown in vitro had larger and more highly vacuolated mesophyll cells than field leaves (unpublished resuits). These observations were confirmed by an increased water content of shoots cultivated in vitro compared with the seedlings (87.5% and 799 respectively). Similar cytological particularities have also been observed in mesophyll cells of adventitious shoots of Picea abies (von Arnold and Eriksson 1984 Seedlings A low NB activity, higher in the roots than in the shoots was detected in seedlings of Pinus pinaster grown in distilled water. The capacity to reduce nltrate was strongly increased when the plants were fed with a KNO~ solution. A higher NR activity has been measured i~ the roots [6.56 ~mol (2 h g d.wt.) -I ] than in the shoots [3.15 ~mol (2 h g d.wt.) -I ] of lodgepole pine (Bigg and Daniel 1978). It is suggested that in Gymnosperms, nitrate is predominantly assimilated in the roots (Smirnoff et al. 1984; Smirnoff and Steward 1985). Nitrate reduction occurred in the roots of 5 week-old non mycorrhizal austrian pines. However, part of the upward nitrogen flux took place as nitrate ions (Martin et al. 1981). Plantlets The potential for the roots of plantlets to develop NR activity in response to nitrate feeding is similar to that of seedlings in the same conditions. The roots of Pinus taeda plantlets and seedlings have been found to have the same ability to take up nitrogen and phos-

371 phorus (McKeand and Allen 1984) when based on an index of root surface area. When the root system of plantlets was dipped in a KNO 3 solution, the shoot exhibited the same level of NR activity as the shoot of seedlings in identical conditions. This result reveals the efficiency of the root-shoot junction. However, we noticed a higher accumulation of nitrate in the aerial part of plantlets compared with seedlings. The reasons stated above (increase of hydric flux, requirement of osmoticum) cannot be used in order to explain the difference since the plantlets have been acclimatized (Wetzstein and Sommer 1982). We suggest that the higher nitrate accumulation in the shoots of plantlets results from a greater uptake by their root system. This hypothesis could be justified by the morphology and the anatomy of the roots. Indeed, at the stage of sampling, the adventitious roots were more heavily branched than the roots of seedlings, with numerous short roots (mycorrhizogenic roots), the simplified structure of which presumably allowed a more efficient uptake of the nutrients (Faye et al. 1981). These data demonstrate that the plants regenerated i__nn vitro and the seedlings in the same situation have an equal capacity to achieve the first step of nitrate assimilation. They also confirm that the differentiation of the root primordia from cells of an axillary shoot is associated with the acquisition of physiological activities of the newly-formed organs. References Aslam M (1981) Plant Physiol 6 8 : 3 0 5 - 3 0 8 Bigg WL, Daniel TW (1978) Plant Soil 5 0 : 3 7 1 - 3 8 5

Bornman C, Vogelmann T (1984) Physiol Plant 61: 505512 Brown CL, Sommer HE (1982) In: Bonga JM, Durzan DJ (eds) Tissue culture in forestry, Nijhoff, Junk, The Hague, pp 109-149 Conner LN, Conner AJ (1984) Plant Sci Lett 3 6 : 2 4 1 - 2 4 6 David A (1982) In: Bonga JM, Durzan DJ (eds) Tissue culture in forestry, Nijhoff, Junk, The Hague, pp 72-108 David H, Isemukali K, David A (1978) C R Ac Sci Paris 287D: 245-248 / Debergh PC (1983) Physiol Plant 5 9 : 2 7 0 - 2 7 6 Faye M, Rancillac M, David A (1981) ~lew Phytol 87: 557-565 Lawrence JM, Herrick HE (1982) Plant Sci Lett 24: 1726 Martin F, Chemardin M, Gadal P (1981) Physiol Plant 53:105-110 McKeand SE, Allen HL (1984) Physiol Plant 6 1 : 5 2 3 - 5 2 8 Rancillac M, Faye M, David A (1982) Physiol Plant 56: 97-101 Robin P, Conejero G, Tranchant JP, Passama L, Salsae L (1983) Physiol Veg 21: 123-128 Smirnoff ~, Todd P, Steward GR (1984) Ann Bot 54: 363374 Smirnoff N, Steward GR (1985) Physiol Plant 64:133-140 Treguer P, Lecorre P (1975) Manuel d'analyses des sels nutritifs dans l'eau de mer, Universit$ de Bretagne occidentale, Brest, France, pp 11-22 yon Arnold S, Eriksson T (1984) Plant Cell Tissue Organ Culture 3 : 2 5 7 - 2 6 4 Wetzstein HY, Sommer HE (1982) Amer J Bot 69: 15791586.

Nitrate reductase activity and nitrate accumulation in in vitro produced axillary shoots, plantlets and seedlings of Pinus pinaster.

The initial and induced in vivo Nitrate Reductase Activity, the nitrate accumulation by in vitro-produced axillary shoots and plantlets of Pinus pinas...
343KB Sizes 0 Downloads 0 Views