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Effect of Nitrogen Over-Supply on Root Structure of Common Reed Author(s): Olga Votrubová and Alena Pecháčková Source: Folia Geobotanica & Phytotaxonomica, Vol. 31, No. 1, Adaptation Strategies in Wetland Plants: Links between Ecology and Physiology. Proceedings of a Workshop (1996), pp. 119- 125+I-II Published by: Springer Stable URL: http://www.jstor.org/stable/4181423 . Accessed: 15/06/2014 22:54 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. . Springer is collaborating with JSTOR to digitize, preserve and extend access to Folia Geobotanica &Phytotaxonomica. http://www.jstor.org This content downloaded from 194.29.185.109 on Sun, 15 Jun 2014 22:54:57 PM All use subject to JSTOR Terms and Conditions

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Effect of Nitrogen Over-Supply on Root Structure of Common ReedAuthor(s): Olga Votrubová and Alena PecháčkováSource: Folia Geobotanica & Phytotaxonomica, Vol. 31, No. 1, Adaptation Strategies in WetlandPlants: Links between Ecology and Physiology. Proceedings of a Workshop (1996), pp. 119-125+I-IIPublished by: SpringerStable URL: http://www.jstor.org/stable/4181423 .

Accessed: 15/06/2014 22:54

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

Springer is collaborating with JSTOR to digitize, preserve and extend access to Folia Geobotanica&Phytotaxonomica.

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Folia Geobot. Phytotax. 31: 119-125, 1996

EFFECT OF NITROGEN OVER-SUPPLY ON ROOT STRUCTURE OF COMMON REED

Olga Votrubova & Alena PechiAkova

Department of Plant Physiology, Faculty of Science, Charles University, Vinic'na 5, CZ-128 44 Prague 2, Czech Republic; tel. +42 2 24915520, fax + 42 2 293643

Keywords: Aerenchyma, Lignification, Phragmnites australis, Root length, Starch deposition

Abstract: Plants of the common reed (Phragmites australis (CAV.) TRIN. ex STEUD.) were treated with high doses of nitrogen in an experiment performed in a sand culture and lasting one vegetation period. Nitrogen treatment resulted in alterations of growth; the increased growth of rhizomes and stalks was connected with production of significantly shorter roots. The differences in root growth were connected with changes in the internal root structure. In rhizome-borne roots, treatment with high nitrogen reduced the formation of aerenchyma and lignified layers in subapical regions. No substantial differences were observed in starch occurrence in the roots under the two treatments.

INTRODUCTION

Anatomical adaptations are necessary for the survival of wetland plants in hypoxic or anoxic substrates. The most important anatomical adaptation is the continuous and extensive system of intercellular spaces, connecting the above-ground plant organs with the below-ground rhizomes and roots, and providing a low resistance pathway for oxygen transport (see e.g. ARMSTRONG & ARMSTRONG 1988, 1990 for details). Additional important anatomical adaptations are barriers against radial oxygen leakage from below-ground organs - the outer layers of rhizomes and of older regions of roots. On the other hand, partial radial oxygen loss from roots to the rhizosphere seems to be advantageous due to the formation of narrow oxygenated zones along the root surfaces which protect the roots against phytotoxic products occurring in anoxic soils (ARMSTRONG 1975). In anoxic substrates, the balance between the two requirements (root tissue respiration and rhizosphere oxidation) is of utmost importance for root survival.

In adventitious roots of reed, intercellular spaces are represented by aerenchyma which is formed in the middle part of root cortex where the cubic and radial arrangement of cells is characteristic of the preaerenchymatous stage of development (JUSTIN & ARMSTRONG 1987, see also Plate 1 a). The radial barrier to oxygen loss in reed roots is represented by one or more layers of tightly packed cells with thickened and lignified cell walls.

It has been suggested that eutrophic substrates, containing large quantities of nutrients (N,P above all) together with high amounts of organic matter and the products of its decomposition may contribute significantly to the reed decline observed in many European habitats during the last few decades (OSTENDORP 1989). Production of insufficient carbohydrate reserves in plants subjected to high nitrogen supply might be responsible for reed die-back as postulated by 0I2KOVA-KON4ALOVA et al. (1992). Results of field measurement relevant to this carbon

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120 0. Votrubova & A. Pechackova

starvation hypothesis are presented in this issue (PINKovA et al. 1996 and KUBiN & MELZER 1996, see also KUBiN 1994).

In addition, changes to the anatomical structures are likely to be evoked by eutrophic conditions. There are several reasons for this assumption: (a) Shoots of reed plants taken from extremely eutrophic habitats showed weak and not well developed sclerenchyma (KLOrzi 1971). (b) Addition of pig farm sewage water to wetland sedges resulted in decreased proportions of aerenchyma in roots, decreased thickening and lignification of the exodermis and lower amounts of starch grains (KONtALOVA et al. 1993). The same treatment of reed plants resulted in decreased root porosity (N2KoVA-KoN0ALovA et al., in press). Changes to the anatomy of wetland plants as described above were observed with plants growing in very complex substrates and it is not possible to distinguish the cause of the changes observed.

With cereals, similar changes were described when plants were treated with high nitrogen doses. Studying causes of cereal lodging, MULDER (1954) observed the reduction of sclerenchymatic tissues and cell wall thickness in stalks of plants subjected to high nitrogen doses. Aerenchyma formation was found to be reduced in maize roots when they were subjected to the simultaneous influence of hypoxia and high nitrate content in the medium (KUBICA & BALUPKA 1988).

Therefore we tested whether aerenchyma formation and lignification in reed roots are influenced by high nitrogen and if so, to what extent, in which regions of the root and at what phase of the vegetation period.

MATERIALS AND METHODS

Cultivation and sampling

The plants were propagated from cuttings of horizontal rhizomes collected from plants cultivated in the greenhouse of the Botanical Institute at Treboni. All cuttings consisted of four nodes and the remains of old stalks and one emerging new stalk about 200 mm high. They were deprived of roots before planting. The cuttings were cultivated in plastic containers (150 x 150 x 170 mm) filled with washed river sand. The cultivation took place outdoors in the Experimental Garden of the Department of Plant Physiology at Prague and lasted from 16 April to 6 October.

The treatments differed as follows. The control treatments received the nutrient solution according to DYKYJOVA & VEBER (1978) with urea and nitrate as nitrogen sources and containing 99 mmol urea 1- and 42 mmol nitrate 1-l supplied as KNO3. The nitrogen enriched treatment was supplied with the same solution but containing fourfold amounts of each nitrogenous compound (396 mmol urea l- and 168 mmol nitrate l-l supplied as a mixture of KNO3, Mg(NO3)2.6H20 and Ca(NO3)2.4H20). The nutrient solutions were changed every two weeks. In the meantime only water was added to maintain water level 10 mm above the sand surface throughout the entire cultivation period.

Plants were harvested at 31 May, 15 June, 30 June, 16 August and 6 October. With every harvest, three control plants and three nitrogen treated plants were used for estimation of the following characteristics: the number of stalks and the length of the highest stalk, length of the rhizome, the number and length of all roots.

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Effect of nitrogen over-supply on root structure 121

Anatomical studies

With respect to the enormous variability in root lengths and types, roots for quantitative anatomical analysis were selected according to following criteria. Only nodal roots emerging from horizontal rhizomes and having the selected length were processed. The selected length was 70 to 100 mm for roots harvested at 19 May and 30 May and 100-150 mm for roots harvested later. At least four roots belonging to various plants were taken.

Ten-millimetre segments were cut out at the root base and at 10 mm, 30 mm, 50 mm and 100 mm distance from the root tip. The segments were fixed in FAA, dehydrated in ethanol and n-butanol series, embedded in paraffin and transversely sectioned at 16 gm. The sections were stained successively in Gentian violet, iodine with potassium iodide and Orange G and mounted into the synthetic resin "Solakryl". Using this procedure, lignified cell walls and starch grauns were stained violet, cellulosic cell walls orange. Lignification was confirmed using phloroglucinol and concentrated hydrochloric acid on transverse hand-cut sections of fixed material. The following anatomical characteristics were evaluated:

(1) Sectional area and percentage of gas space using the point-counting method (WEIBEL 1979);

(2) Number of lignified cell layers in the outer cortex; (3) Occurrence of starch grains.

RESULTS

The growth of the reed plants was influenced considerably by nitrogen enrichment of medium (Tab. 1). Almost all parameters increased as a consequence of the nitrogen enrichment during the whole vegetation period with the exception of roots. The slightly slower initial growth of roots of nitrogen treated plants was accelerated later in ontogeny resulting in the increased total length of roots per plant. On the other hand, the mean root length remained significantly lower, as high nitrogen content evoked increased production of very short roots (to 30 mm), especially towards the end of the vegetation period. In October, these roots

Table 1 . Growth characteristics of reed plants as affected by nitrogen treatment (means of 3 plants). C - control, N - nitrogen treatment. Asterisks indicate differences between treatments significant at the 0.05 probability level. Using t-test.

Variant Date of Length of the Stalk Length of the new Mean length of Root sampling highest stalk (mm) number rhizome (mm) the root (mm) number

C 16 April 200 1 0 0 0 31 May 275 2 0 54 8.7 15 June 320 5 0 78 21 30 June 360 5 225 53 45

16 August 500 7 330 109 66* 6 October 485 1 1 876 93 95*

N 16 April 200 1 0 0 0 31 May 216 3 0 50.6 7.7 15 June 245 8 32 59 39 30 June 430 9 390 59 76

16 August 710 18 1100 60.3 163* 6 October 920 20 1790 68.5 223*

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122 0. Votrubovc & A. Pechc6kovc

C

1 40 s

I I | | | | ,~~~~~~~ 10m

30mmv

20 20

40 .~~ ~ ~~~~~~~~~ lo .

31.5 15.6 30.6 16.8 6.10 30m

20

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31.5 15.6 30.8 18.8 6.10

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Fig. 1.The influence of nitrogen treatmrent on gas space formation in reed roots. The proportion of gas space is expressed as a percentage of the total section area as dependent on time of harvest at 10 and 30 mm from the root tip, above - control (C), below - nitrogen treatmfent (N).

represented 35% of the total number of roots in high nitrogen treated plants compared to 13.5% in control plants. On the other hand, roots longer than 200 mm represented only 3.5% in nitrogen treated plants but 9% in control plants.

T'he internal root structure was not much changed in the older parts of roots where no differences in gas space proportion and appearance were found; both types were as in Plate Ic. The proportion of gas spaces reached as much as 65% of the total sectional area. In contrast, differences were found in subapical parts of roots (Fig. 1). The proportion of gas spaces changed with time in both treatments and was negatively affected by high nitrogen treatment.

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Effect of nitrogen over-supply on root structure 123

Table 2. Occurrence of lignified layers in outer cortex as affected by nitrogen treatment. C - control, N - nitrogen treatment.

Variant Distance of cross Mean number of lignified cell layers (n = 4) section from the

root tip (mm) 31 May 15 June 30 June 16 August 6 October

C 10 I 0 0.5 0.5 3 100 1 1 3 5 1

N 10 0 0 0 0 0 100 0 2 0.5 3 3

The development of lignified cell layers in the outer cortex is documented in Tab. 2. In the older parts of roots, the slight decrease in the number of lignified cell layers in nitrogen treated roots is not significant due to substantial variability in their number. Again, the most conspicuous differences were found near the root tip (at 10 mm) where lignified layers were never observed in nitrogen treated roots in contrast to the controls. As demonstrated in Plate 2a,c, the discontinuity of lignified layers opposite the growing root primordia as described by JUSTIN & ARMSTRONG (1987) was confirmed.

Starch grains (Plate Ic and 2a) were recorded at first in the basal region of roots relatively late in the vegetation period (16 August). Later their formation proceeded towards the root tip and at the end of the vegetation period they were present in all root parts examined. No substantial differences in starch grain appearance and their distribution were found between the two treatments. However, in nitrogen treated roots the progress of starch grains towards the root tip was slightly slower than in control roots. The starch grains were never detected in very short roots (less than 30 mm) but the relation between root length and starch grain occurrence was not studied systematically.

DISCUSSION

The positive effect of high nitrogen content on stalk and rhizome growth was connected with considerable changes in the arrangement of root system. The changes in root system developed gradually in the course of the vegetation period. Whilst at the beginning of the vegetation period the frequency of roots belonging to differing length categories were similar in the two treatments, the situation changed considerably later (16 August and especially 6 October), when the production of short roots was stimulated by high nitrogen content. In addition, further differences are suspected, namely in the distribution of stalks, which seem to be more clustered, in rhizome branching, etc. The number of observations made up until now is however insufficient to make serious conclusions. We assume that not only the inner structure, but also the distribution and the mutual position of sites of oxygen intake (living stalks) and sites of oxygen consumption (rhizomes and roots) are important for oxygen availability in various parts of the plants. Therefore, more detailed studies of general morphology might be necessary.

The development of the inner structure of roots was studied with only one type of root as it is known that maturation position of root tissues is highly dependent on root length as well as on rate of growth (RoST 1994). At the beginning of the cultivation period a low proportion

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124 0. Votrubova & A. Pechackova

of gas spaces was found near the root apex in both treatments (Fig. 1). This coincided with a period of rapid growth of a relatively small amount of new roots. It could be said that the roots produced at the beginning of the experiment had approximately the same history, as they were all newly formed roots emerging from old rhizomes and had approximately the same length. We assume that the growth rate of the roots studied was high in this period and affected the gas space differentiation more than the nitrogen surplus. The period of low proportion of gas spaces was relatively short in roots of control plants but much longer in roots of nitrogen treated plants. In addition, the increase of gas space proportion towards the end of the cultivation period, though observed in both treatments, was much less pronounced in nitrogen treated roots than in the controls. It is likely that in this period nitrogen surplus was the main cause of the differences observed. Nevertheless, it must be emphasized that we could not be sure of the identical history of these roots (i.e. age, growth rate, etc.) as the variation of root lengths was large, new roots were arising up to the end of the vegetation period and the procedure of estimation of root characteristics was destructive.

The development of lignified cell layers was influenced in a similar way. Again it is likely that the onset of their development is affected by nitrogen surplus together with the rate of growth rate of the roots. In nitrogen treated roots the lignified layers were never observed at 10 mm distance from the root apex while in control roots of comparable length the lignified cell layers were normally present in this region. They were not recorded only at 15 June which coincided with the minimum value in gas space proportion. It can be concluded that, as in the case of gas spaces, the main reason is the rapid growth of newly formed roots in this period.

The conspicuous differences in the proportion of gas spaces and lignification of outer regions of roots were recorded previously for wetland sedges after treatment with nitrogen-rich sewage water (KONWALovA et al. 1993 and 6I2KOVA-KONWALOvA et al., in press). In comparison the differences observed in reed roots in response to nitrogen enrichment were rather slight and only thorough quantitative analysis detected any variations. It is likely that not the high nitrogen alone, but the high nitrogen combined with organic matter was responsible for the striking changes observed in wetland sedge roots. This seems to be confirmed by the work of if2KOVA-KONWALOVA & BAUER (1993) showing the different changes in the amount of root aerenchyma evoked by high nitrogen itself and by nitrogen in combination with organic matter.

No striking changes in starch grain appearance were found, whereas in Carex roots treated with sewage water the starch grains were rare or even absent. Nor did the patterns of distribution of starch grains differ in the two treatments. Compound starch grains were always observed in radial cortical cell layers between aerenchyma channels in the lignified outer cortex, in the endodermis and in stelar parenchyma (Plate 1, 2). In contrast to rhizomes where starch is present during the whole year (KUBiN 1994), in roots it is not formed before late summer. In view of the observed absence of starch in short roots, the number of which is much higher in nitrogen treated plants, work is in progress testing starch grain occurrence as dependent on root length.

It can be concluded that high amounts of nitrogen evoked the presumed changes in root structure together with changes in the general morphology of plants. The anatomical structures which were found to be changed are known to influence the oxygen budget in roots. High

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Effect of nitrogen over-supply on root structure 125

nitrogen supply can be considered as one of the factors contributing to structural changes observed in eutrophic habitats.

Acknowledgements: The work was supported by a grant from Charles University, Prague, Czech Republic.

REFERENCES

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ARMSTRONG J. & ARMSTRONG W. (1990): Pathways and mechanisms of oxygen transport in Phragmites australis. In: COOPER P.F. & FINDLATER B.C. (eds.), Constructed wetlands in water pollution control, Pergamon Press. pp. 529-533.

ARMSTRONG W. (I1975): Waterlogged soils. In: ETHERINGTON J.R. (ed.), Environment and plant ecology, John Wiley & Sons, London, pp. 181-218.

CiZKOVA-KONCALOVA H. & BAUER V. (1993): Response of a wetland sedge, Carex gracilis CURT., to hyper-cutrophic conditions: Interactions between anaerobiosis and high nitrogen availibility. Limnol. Aktuell 5: 23-32.

CfZKOVA-KONCALoVA H., KvET J. & LUKAVSKA J. (in press): Response of Phragmites australis, Glyceria aquatica and Typha latifolia to addition of piggery sewage in a flooded sand culture. Wetlands Ecol. Managem.

CIZKOVA-KONCALOVA H., KVET J. & THOMPSON K. (1992): Carbon starvation - a key to reed decline in eutrophic lakes. Aquatic Bot. 43: 105-113.

CIZKOVA H, LUKAVSKA J., PRIBAN K., KOPECKY J. & BRABCOVA H. (1966): Carbohydrate levels in rhizomes of Phragmites australis at an oligotrophic and a eutrophic sites: a preliminary study. Folia Geobot. Phytotax. 31: 111-118.

DYKYJOVA D. & HRADECKA D. (1976): Production ecology of Phragmites communis 1. Relations of two ecotypes to the microclimate and nutrient conditions of habitat. Folia Geobot. Phytotax. 11: 23-61.

DYKYJOVA D. & VEBER K. (1978): Experimental hydroponic cultivation of helophytes. In: DYKYJOVA D. & KVET J. (eds.), Pond littoral ecosystems, Springer Verlag, Berlin, pp. 181-192.

JUSTIN S.H.FW. & ARMSTRONG W. (1987): The anatomical characteristics of roots and plant response to soil flooding. New Phytol. 106: 465-495.

KLOTZLI F. (1971): Biogenous influence on aquatic macrophytes, especially Phragmites communis. Hidrobiologia 12: 107-1 1 1.

KONCALOVA H., KVET J., POKORNY J. & HAUSER V. (1993): Effect of flooding with sewage water on hree wetland sedges. Wetlands Ecol. Managem. 2: 199-21 1.

KUBICA S. & BALUSKA F. (1988): Maize primary root growth and differentiation under conditions of nitrate over-supply. Biolkgia (Bratislava) 44: 407-414.

KUBiN P. (1994): Zmeny obsahu zdsobnich sacharidu mokradnich rostlin na oligotrofnich a eutrofnich stanovi?t[ch (Changes of reserve carbohydrate content in wetland plants from oligotrophic and eutrophic habitats). PhD. Thesis, Charles University, Prague.

KUBiN P. & MELZER A. (1996): Does ammonium affect accumulation of starch in rhizomes of Phragmites australis (CAV.) TRIN. ex STEUD.? Folia Geobot. Phytotax. 31: 99-109.

ROST T.L. (1994): Root tip organization and the spatial relationships of differentiation events. In: IQBAL M. (ed.), Growth patterns in vascular plants. Dioscorides Press, Portland, Oregon.

MULDER E.G. (1954): Effect of mineral nutrition on lodging of cereals. Pl. & Soil 5: 246-306. WEIBEL E.R. (1979): Stereological methods. Practical methods for biological morphometry. Acadermic Press,

London.

Encl. Plate 1, 2, pp. I-II.

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0. Votrubovd & A. Pechdckovd: Effect of nitrogen over-supply on root structure

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Plate 1. Transverse sections of reed roots harvested at 6 October; a - 5 mm from root tip, b - 10 mm from root tip, c - root base, 1 - root cortex with cells in radial and cubical arrangement, preaerechymatous stage of development, 2 - beginning of aerenchyma formation (b), fully developed aerenchyma (c), 3 - barriers against oxygen leakage in outer cortex, beginning of formation (b), fully developed (c), 4 - cortical cells with starch grains, 5 - fully developed endodermis, bar indicates 600 pm. (All sections shown in Plate 1 were taken from control roots, the structures of nitrogen treated roots did not differ substantially, the differences were only quantitative.)

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11 0. Votrubovi & A. Pechdckovd: Effect of nitrogen over-supply on root structure

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Plate 2. Transverse sections of reed roots harvested 16 August (b,c) and 6 October (a); a - detail of cortical parenchyma at root base showing compound starch grains, b,c - transverse sections at 50 mm from tip with lateral root primordium showing no aerenchyma and no thickened layers opposite root primordium, 1 - ligni- fled layers, 2 - root primordium, 3 - cortical cells with schizogenous intercellular spaces, 4 - cortical cells between aerenchyma channels, 5 - compound starch grain; bars indicate 60 pm (a), 240 pm (b), and 600 pm (c). (All sections shown in Plate 2 were taken from control roots, the structures of nitrogen treated roots did not differ substantially, the differences were only quantitative.)

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