Shoot-root interaction effects on nitrate reductase and glutamine synthetase activities in rose (Rosa × hybrida cvs. Ilseta and Mercedes) graftlings

J Plant Physiol. VtJL

149. pp. 559-563 (1996)

Shoot-Root Interaction Effects on Nitrate Reductase and Glutamine Synthetase Activities in Rose (Rosa x hybrida CVS. IIseta and Mercedes) Graftlings 1 2 HASAN AGBARIA , BRURIA HEUER , 1

2

and

NAFTALY ZIESLIN

1

*

The Kennedy-Leigh Center for Horticulture Research, Faculry of Agriculture, The Hebrew Universiry of Jerusalem, P.O. Box 12, Rehovot 76-100, Israel Institute of Soils and Water, ARO, The Volcani Center, Bet Dagan 50-250, Israel

Received November 20, 1995 . Accepted March 5, 1996

Summary

Flower formation in greenhouse roses (Rosa x hybrida) is generally promoted by grafting on various rootstocks rather than propagating ungrafted plants from cuttings. The number of flowers per plant in roses is also positively correlated with the nitrogen content in the nutrient solutions as well as by ammonium ion proportion of the total nitrogen. It could be, therefore, that the differences among the effects of various root systems on flower formation in rose plants are associated with differences in assimilation of nitrogen, in either nitrate or ammonium-forms or both. This assumption was examined by measurements of the nitrate content as well as of the nitrate reductase (NR) (E.C. 1.6.6.1) and glutamine synthetase (GS) (E.C. 6.3.1.2) activities in roots and leaves of 6-week-old plants propagated from cuttings (own-root plants) or graftlings, i.e., concomitantly grafted and rooted roses cvs. Mercedes, Ilseta or Rosa indica major, used as understocks. The data obtained showed that the nitrate contents in the roots of cuttings were higher than those in the leaves, whereas that in the roots of R. indica was higher than those in roots of the two cultivars. The content ofN03 - in roots and leaves of the graftlings was affected by the grafting procedure and was in good agreement with the nitrate reductase activity in plant organs. The activity of nitrate reductase in the leaves of cuttings was higher than in their roots, whereas in graftlings, its activity was promoted in the roots and inhibited in the leaves, in all scion-root combinations. Glutamine synthetase activity in roots and leaves of either cuttings or graftlings was 4-5 orders of magnitude higher than NR activity. Similar to NR activity, GS activity in the leaves of own-root plants was higher than that in their roots. However, unlike the NR activity, GS activity in roots and leaves of the graftlings was not markedly altered following the grafting procedure.

Key words: Nitrate reductase, glutamine synthetase, shoot-root interactions, grafting, roses, Rosa hybrida. Abbreviations: NR = nitrate reductase; GS = glutamine synthetase; GAM = glutamic acid monohydro-

xymate. Introduction

Greenhouse rose plants grown in controlled environments are characterized by recurrent, year-round vegetative growth of lateral shoots with terminal flowers (Zieslin and Moe,

* Correspondence. © 1996 by Gustav Fischer Verlag, Stuttgart

1985). Hence, no separation can be made between the vegetative and reproductive phases of plant development and, in contrast to numerous other plant species, these two phases are not antagonistic. Accordingly, it was shown (Bik, 1972) that promotion of vegetative growth in rose plants was accompanied by increased flower formation and that the increase was positively correlated with the level of nitrogen

560

HASAN AGBARIA, BRURIA HEUER, and NAFTALY ZIESLIN

nutrition. It has also been found (Feigin et al., 1986; White and Richter, 1973) that rose flower formation was promoted by incorporation of ammonium ions into nutrient solutions as part of the nitrogen nutrition and that root formation of rose plandets in vitro was stimulated by addition of ammonium ions (Hyndman et al., 1982). It is also known that the number of flowers of rose (Rosa X hybrida) plants grafted on various selected rootstocks is generally higher than that of ungrafted plants from rooted cuttings, especially when planted in soils or soil mixtures (Zieslin and Snir, 1989). The presence of compounds in calli of rose plants, capable of promoting growth of rose explants, has been demonstrated in vitro (Zieslin et al., 1987). However, morphological and physiological aspects of flower formation, promoting effects of rootstocks in rose plants with recurrent blooming, has seldom been investigsted and only scarcely reported. The two reported phenomena, promotive effects of nitrogen nutrition on flower formation and the positive effects of various rootstocks on flower formation in rose plants, suggest that the effects of the rootstocks in rose plants might be associated with differences among root systems in their ability to assimilate nitrogen, either as nitrate N0 3 - or ammonium NH4 + ions, or both. It seems resonable to assume that the differences in assimilation of nitrogen ions should be expressed by the differences in activities of two key enzymes involved in the nitrogen metabolism in plants: nitrate reductase (NR) and glutamine synthetase (GS). Therefore, the first step in the investigation of the possible association between the effects of rootstocks on flower development and nitrogen metabolism was to determine the activities of these enzymes in roots and shoots of various scion-root combinations of several-week-old graftlings. Materials and Methods

Plant material and propagation procedure Graftlings, i.e., concomitantly grafted and rooted cuttings (van de Pol and Breukelaar, 1982) of rose (Rosa x hybrida) cvs. I1seta and Mercedes and of Rosa indica major, a known rose rootstock, were used in the various experiments. Homografts of on Rosa indica were used as the scion-root combinations of the gtafts. Ungrafted rooted cuttings (own-root plants) of cvs. Mercedes, I1seta and R indica, as well as autografts of Mercedes on Mercedes and I1seta on I1seta, were employed as the control treatments. The various scion-root combinations were obtained by the T-budding method (Chu, 1990), by inserting an axiallary bud of the scion beneath a leaf of the understock. The cuttings or graftlings, with the base dipped into rooting powder containing 0.8 % indolebutyric acid, were inserted into rockwool cubes (4 x 4 x 4 cm) and were placed inside aeroponic foggers (Shira Airoponics, Rehovot, Israel) illustrated elsewhere (Zieslin and Abolitz, 1994). The foggers were located in a greenhouse where minimum and maximum temperatures of 18 and 28 'C, respectively, were continuously maintained. Tap water, with an electrical conductance of 0.6 dS . m -1, containing trace amounts of nitrates, was used for generation of fog during the rooting period. The 5 - 8 cm of the distal parts of the roots protruding from the rockwool cubes, or the leaves of the newly formed shoots of 4-5-week-old plants were used for measuring nitrate content and the enzymatic activity. For the comparative analysis of the

scion-root interaction effects, the tissues of the various scion-root combinations and those of the control treatments were always concomitantly collected at noon time and stored according to the established procedures.

Nitrate content A 100-mg sample was dispersed for 60 min in double-distilled water at 45 'c. The suspension was centrifuged for 15 min at 6500 Kn (Sorvall, RC-285) and 200 ~L of the supernatant were incubated for 20 min at room temperature with 0.8 mL 5 % salicylic acid in concentrated H 2S04, Following addition of 19 mL 2N NaOH for pH adjustment to 12, the samples were cooled to ambient temperature and the coloration developed was measured spectrophotometrically (Varian OMS-IOO) at A = 410 nm. The nitrate content was calculated according to a standard curve obtained from solutions with known concentrations ofKN0 3 (Cataldo et al., 1975).

Nitrate reductase (NR, NADH: nitrate oxiMreductase, EC 1.661) Both in vitro and in vivo methods of NR assays were examined in preliminary experiments. In contrast to the in vivo assays, the results of the in vitro method for determining NR activity in rose tissues were erratic and inconsistent. The in vivo assay was, therefore, used during the study. The NR activity was measured in leaf discs, 9 mm in diameter, or in a weight-equivalent amount of roots, according to Heuer and Plaut (1978). The tissue samples were infiltrated under vacuum at 0-2 'C in 10 mL 50 mmol/L phosphate buffer, pH 7.5, containing 0.1 mol/L KN0 3, and 0.1 % Triton X-lOO. After 5 min, the tissue samples were transferred into an identical buffer solution but without Triton X-IOO and incubated for 1 hat 28 'c. For determination of the nitrite formed, 1 mL of the solution was supplemented with 0.25 mL of 1.5 mol/L HCI containing (w/v) 1 % sulfanilamide and 0.25 mL of a 0.02 % solution (w/v) of N-(l-naphthyl(ethylenediamine) dihydrochloride. The absorbance of the coloration formed was measured at 540 nm and the NR activity was calculated from a standard curve ofKN0 2 .

Glutamine synthetase (GS, EC 63.1.2) Glutamine synthetase activity was examined by quantitative measurements of L-glutamic acid monohydroxymate formed from glutamic acid by the action of GS (Brun et al., 1992). A 1 -g sample of leaf or root tissue was crushed in 10 mL 50 mM Tris-HCl buffer, pH 7.6, containing 2 % (w/v) PVP-45, 10 % insoluble PVPp, 10 0/0 glycerol, 2 mM EOTA, 14 mM ~-mercaptoethanol, 5 mM MgS04 and 10 mM glutamate, at 0-2 'c. Following filtration of the crushed tissue through four layers of cheesecloth, the filtrate was homogenized with a glass-teflon rod (Elda, Israel) and centrifuged for 30 min at 40,000 Kn. For determination of the enzymatic activity, 0.1 mL of supernatant was supplemented with 1 mL 5 mmol/L trisHCl buffer, pH 7.2, which contained 30 mmol/L NHzOH, 20 mmol/L MgS04' 20 mmol/L arsenate, 4 mmollL EOTA, 0.5 mmollL ADP and 125 mmollL glutamine. The reaction was terminated after 30 min by addition of 1 mL 6 M HCI with 0.37 M FeCI, and 40 % (w/v) TCA. Following 10 min cooling, absorbance was measured at 540 nm. The activity of the enzyme was calculated from a standard absorption curve of y-glutamyl hydroxamate (GAM) formed from glutamic acid and the reaction mixture.

Statistical analysis For statistical analysis, shoot or root tissues collected from individual plants were combined. Following mixing, the measurements in each treatment were repeated nine times.

Shoot-Root Interaction Effects on Nitrate Reductase and Glutamine Synthetase Activities in Rose

Results and Discussion

The content of free nitrate in roots of cv. Mercedes (Table 1) and of those of cv. IIseta propagated from cutting was much higher than in their leaves, by 53 and 95 %, respectively, suggesting that enhanced enzymatic activity occurs in the leaves. In roots of R. indica cuttings, the nitrate content was twice that in roots of cv. Mercedes and 62 % hi§her than in roots of cv. I!seta, 27.2., 13.2 and 16.8 mggFW- , respectively. The nitrate in roots and leaves of young (4-6 weeks old), non-fertilized, aeroponically rooted rose cuttings probably originated from the propagation material and from the traces of nitrate found in the water used for fog generation. The higher content of N0 3 - in the roots than in the leaves could derive from higher density of the nitrate-containing fog around the roots. Higher activity of nitrate reductase (NR) in leaves of own-root plants of cvs. Mercedes and IIseta than in their roots supported this assumption. It was found (Table 2) that the NR activities in the leaves of these two cultivars were 2.5 and 3.5 times respectively higher, than those in the roots. These results are in good agreement with higher rates of nitrate reduction in leaves than in roots reported in other plants (Aslam and Huffaker, 1982). The activity of NR in roots of R. indica was similar to that in the roots of cv. Mercedes and by 75 % higher than in the roots of cv. 11seta (Table 2). The nitrate content in roots of cvs. Mercedes and IIseta autografts and reciprocal homografts was higher than the content in roots of non-grafted plants of these cultivars (Table 3). On the other hand, the nitrate content in heterograft roots of R. indica was reduced following grafting with scions of cv. Mercedes or cv. IIseta (Table 3). Elevated levels ofN03 - were also found in leaves of cv. Mercedes graftlings, whereas in leaves of cv. IIseta homografts on Mercedes as an understock, the nitrate content was lower than in leaves

561

Table 3: Nitrate content (mg N0 3 - g F . W- I ) in roots and leaves of grafts of rose (Rosa x hybrida) cvs. Mercedes and Ilseta on , or Rosa indica major as understocks. Values are means of 9 replicates. Understock

Nitrate content Mercedes

Mercedes Ilseta

R. indica

LSDoo5

Ilseta

Roots

Leaves

Roots

Leaves

16.3 25.3 17.2 3.59

13.2 10.9 12.2 0.66

15.0 24.1 20.9 8.15

6.2 10.9 16.8 0.64

Table 4: Nitrate reductase (NR) activity (nmol N0 2 - g FW-I h -I) in roots and leaves of grafts of rose (Rosa x hybrida) cvs. Mercedes and Ilseta on , or Rosa indica major. as understocks. Values are means of 9 replicates. Understock

NR activity Mercedes

Mercedes Ilseta

R. indica

LSDo.05

Ilseta

Roots

Leaves

Roots

Leaves

94.5 63.0 111.3 5.77

56.5 94.1 78.6 17.23

94.1 96.9 103.9 4.73

54.2 64.9 95.3 21.81

of own-root plants. Similar to the own-roots plants (Table 1), the content of free nitrate in the roots of graftlings was also higher than that in their leaves (Table 3). However, the alterations in nitrate content imposed by the grafting procedure and the type of the understock as well as the ratio in N03 content between the roots and the leaves varied in the differTable 1: Nitrate content (mg N0 3- g F . W-1) in roots and leaves of ent scion-root combinations (Table 3). rooted cuttings of rose (Rosa x hybrida) cvs. Mercedes, Ilseta and The reasons for the grafting-imposed increases in the Rosa indica major. Values are mean of9 replicates. N0 3 - content in roots and leaves of cvs. Mercedes and IIseta autografts or homografts, and for the decreases in the N0 3 Cultivar Nitrate content contents in the roots of R. indica bearing either cv. Mercedes Leaves or cv. IIseta scions, are still not clear. We consider that the Roots variations stem from grafting-imposed alterations in enzyMercedes 13.2 8.6 matic activity of the nitrogen metabolism and not from disIlseta 16.8 8.6 ruption of the vascular connection between the understocks R. indica 27.2 and scions, as has been reported for other plants (Andrews 1.64 0.8 LSDo.05 and Marquez, 1993), including roses (Marcelis-van Acker, 1993). Similar to the increase in nitrate content, remarkable alterTable 2: Nitrate reductase (NR) activity (nmol N0 2 - g FW- I h -I) in ations in NR activity in the roots of all three rose types used roots and leaves of rooted cuttings of rose (Rosa x hybrida) cvs. Mer- as understocks were observed following the grafting procecedes, Ilseta or Rosa indica major. Values are means of 9 replicates. dure (Table 4). The most pronounced effect was a 2.8-fold increase in the NR activity measured in roots of the cv. IIseta Cultivar NRactivity autografts, whereas the increase in NR activity in roots Leaves with cv. Mercedes as a scion was only 84 %. Roots Nitrate reductase is considered to be an enzyme induced Mercedes 140.6 55.3 by the presence of N0 3 - and its activity is correlated with Ilseta 121.6 34.2 the amount of the nitrate that serves as a substrate (CampR. indica 59.8 bell, 1988; Campbell and Smarrelli, 1986; Jackson et al., 3.14 LSDo.o5 15.34 1986). The increased NR activity in roots of Mercedes and

562

HASAN AGBARIA, BRURIA HEUER, and NAFTALY ZIESLIN

Ilseta understocks partly coincided with increased N0 3 contents, although not to the same extent, while in R. indica it resulted in rather low contents of nitrate. The elevated NR activity in roots of R. indica may result from a higher level of enzyme synthesis, due to reduced turnover of the enzyme or from a higher availability of additional factors, such as NADH and NAD(P)H, involved in the regulation ofNR activity (Campbell and Smarrelli, 1986; Oaks and Hirel, 1985). All of these hypotheses still require further investigation. In contrast to its effect on the roots, the grafting resulted in a remarkable decrease in NR activity in the leaves of the graftlings (Table 4) as compared with the activity in the leaves of own-root plants (Table 2). It has been previously reported (Andrews and Marquez, 1993) that the activity of various enzymes in the leaves of fruit trees was inhibited after grafting. This could also be the case for NR activity in the leaves of rose graftlings. The observed differences between the effects of grafting on NR activity in roots and leaves may also indicate that the NR of leaves differs from that of the roots, as was previously suggested by Oaks and Hirel (1985). The results of several studies (Feigin et al., 1986; Laurie and Kiplinger, 1944; White and Richter, 1973) showed that growth and flower formation in rose plants were promoted when ammonium ions were incorporated into the nitrogen nutrition. A major route for assimilation of ammonium ions either in the roots or in the leaves is represented by the function of glutamine synthetase (GS) (Oaks and Hirel, 1985). Therefore, the activity of this enzyme was also examined in the present study. The levels of GS activity in roots and leaves of own-root plants (Table 5) were 2 and 2.5 times, greater, respectively, than those ofNR activity (Table 2). Yet, similar to the NR activity, the GS activity in the leaves was considerably higher than that in the roots and the activity of GS in the roots of R. indica was higher than that in the roots of the other two cultivars. Contrarily, the data also showed that the GS activity in the roots of all three cultivars examined was only little affected by the grafting procedure and by the cultivar of the scion (Table 6). It was previously shown (Zieslin and Snir, 1989) that the capacity for acidification in roots of R. indica was higher than that of roots of the other rose cultivars. If we assume that the acidification capacity is partly due to enhanced ammonium assimilation, then it may be possible to associate it with the higher GS activity in the roots of this rose species, widely used as rootstocks, especially in areas with calcareous soils. The higher GS activity in roots of non-grafted plants of R.

Table 6: Glutamine synthetase (GS) activity (Jlmol GAM g FW- I

h -I) in roots and leaves of graftlings of rose (Rosa x hybrida) cvs. Mercedes and I1seta on Mercedes, I1seta or Rosa indica major as understocks. Values are means of9 replicates.

h- I ) in roots and leaves of rooted cuttings of rose (Rosa x hybrida) cvs. Mercedes, I1seta and Rosa indica major. Values are means of 9 replicates. GS activity

Cultivar

Mercedes Iiseta

R. indica LSDo.05

Roots

Leaves

101.4 119.6 198.3 4.94

304.4 300.4 8.56

I1seta

Mercedes

Mercedes I1seta

R. indica LSD o.05

Roots

Leaves

Roots

Leaves

107.6 111.3 204.1 7.44

350.0 306.2 528.6 18.26

99.9 124.5 249.8 3.21

264.7 372.9 520.3 19.27

indica than in roots of non-grafted plants of the other two cultivars, (Table 5) as well as the higher GS activity in R. indica roots of the cv. Mercedes and Ilseta heterografts compared with the other two cultivars used as understocks (Table 6), support this assumption. The enhanced GS activity in leaves of the scions grafted on R. indica is additional evidence for this assumption. However, the mode how this promotion in the enzymatic activity is obtained still requires further investigation. To the best of our knowledge, the question of the alteration of NR and GS activities in plant roots and leaves by various scion-root grafting combinations has not yet been addressed, although the functions of these two enzymes and the regulation of their activities have been extensively studied, described and reviewed (Campbell and Smarrelli, 1986; Lillo, 1994; Mohr et al., 1992; Solomonson and Barber, 1986). However, it is possible that the findings of the present study reflect a situation that may be present only in young plants, which are deprived of nutrients and exposed to low light intensities and, therefore, have underdeveloped and not-yet stabilized functions, whereas the effects of the scion-root interaction on the activity of enzymes involved in nitrogen assimilation may differ in older, more developed plants, cultivated in more favorable conditions. For the same reason, it is still premature to correlate plant growth and flower formation with the relationships between enzyme activities and scionroot interactions observed in the young plantlets. This is also true of the possible use of these activities for selection of own-root rose plants or better understocks. References

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Table 5: Glutamine synthetase (GS) activity (Jlmol GAM g FW- 1

GS activity

Understock

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