In situ assay of nitrate reductase activity using ...

Environ Monit Assess (2017) 189:332 DOI 10.1007/s10661-017-6045-9

In situ assay of nitrate reductase activity using portable water bath Adam Rajsz & Bronisław Wojtuń & Andrzej Bytnerowicz

Received: 19 February 2017 / Accepted: 30 May 2017 # Springer International Publishing Switzerland 2017

Abstract In environmental research (i.e., plant ecophysiology, environmental microbiology, and environmental chemistry), some assays require incubation of samples at controlled temperature and darkness. Until now, due to a lack of equipment providing such possibility in situ, researchers had to move collected samples to the laboratory for incubation. Obviously, a delayed incubation and the ex situ conditions could seriously affect the assays’ results. A good example of analysis where water bath use is needed is the nitrate reductase activity (NRA) in vivo assay where plant tissue samples are incubated in buffer solution at a predetermined temperature. We designed a transportable water bath with a temperature control which enables in situ measurements in many types of environmental studies. The presented device is small in size featuring a thermally insulated chamber and an electronically controlled thermostat system powered by a 12-V battery. Due to its modular design, it can be transported comfortably in difficult terrain. The incubation process can be carried out continuously in stable temperature and darkness. In order to examine the field usability of the presented device, we conducted measurements of plant nitrate reductase A. Rajsz (*) : B. Wojtuń Department of Ecology, Biogeochemistry and Environmental Protection, Wrocław University, Kanonia 6/8, 50-328, Wroclaw, Poland e-mail: [email protected] A. Bytnerowicz USDA Forest Service, Pacific Southwest Research Station, 4955 Canyon Crest Drive, Riverside, CA 92507, USA

activity in difficult field conditions. The in situ assays were carried out at high altitudes in the Karkonosze mountains, SW Poland. The NRA was studied in two alpine species (Deschampsia caespitosa and Homogyne alpina). Our results showed low NR activity in H. alpina (mean 0.31 μM NO2 g−1 DW h−1) and higher NRA in D. caespitosa (mean 2.7 μM NO2 g−1 DW h−1). The obtained results were highly reproducible and had small variability (low standard error values). Keywords Field analyses . Transportable water bath . Incubation . In vivo method . Nitrate reductase activity (NRA) . In situ conditions

Introduction Research techniques in modern biology are focused on enabling observation, measurements, and determination of physical and biochemical parameters in vivo with the least possible disturbance in a functioning of living system in their natural environment. Due to the fact that plants cannot change their location, all vital parameters are strongly linked to their external conditions. Because of this fact, plants are often used for monitoring of environment conditions. Thus, in the ecophysiological and environmental studies, a possibility of measurement conducted directly under field conditions is very important. The in situ methods in biology inevitably encounter many obstacles particularly in areas difficult to access for humans such as alpine, polar, wetland, or desert ecosystems. It becomes necessary not only to develop

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new methods that allow to carry out analyses and measurements quickly, conveniently, and inexpensively but there is also an increasing need for equipment which could simplify and improve field measurements and their accuracy. One such limitation is the inability to use a laboratory water bath for incubation of samples for some chemical, physiological, or microbiological analyses performed in situ in difficult-to-access terrain. A good example of a field assay where incubation of samples in controlled temperature and darkness provided by a water bath is needed is a plant nitrate reductase activity (NRA) assay conducted in situ. Nitrate reductase (NR) is one of the most intensively studied plant enzymes involved in primary nitrogen metabolism. NRA analysis in situ is a widely used method in plant ecophysiological studies (Harper and Hageman 1972; Norby 1989; Norby et al. 1989; Calatayud et al. 2007; Tang et al. 2012; Dovis et al. 2014). There is also growing interest in NRA assays conducted in situ in order to capture as accurately as possible environmental variables affecting plant physiological parameters in the field conditions (Krywult and Bielec 2013; Tang et al. 2012). The study of plant NRA can be useful for the evaluation of many ecological problems. Some authors use it for the assessment of plants’ response to anthropogenic nitrogen pollution of the air (Krywult and Bytnerowicz 1997; Diekmann and Falkengren-Grerup 2002) or for revealing mechanisms of biological invasions (Filippou et al. 2014; Chmura et al. 2016). It can be also used for population studies (Sakar et al. 2010). Recently, NRA has also started to be used as one of the useful plant functional traits (Arslan and Güleryüz 2005;Dias et al. 2011) due to the growing evidence that functional traits rather than species are a better tool to analyze how an ecosystem works (McGill et al. 2006). Our improved field application of the discussed method can be valuable for environmental research where measurements should be performed in undisturbed environmental conditions (i.e., air temperature, humidity, actual insolation, water status, and overall plant energy balance). These variables are unstable by their very nature. For this purpose, it becomes necessary to use a water bath for sample incubation under specific field conditions. Laboratory water baths are heavy, bulky devices with an AC power supply and are not suitable for outdoor use. Some researchers working on NRA assay have attempted to move entire plants to the laboratory and pot them (Krywult et al. 2002), while others have moved collected fragments of living plant tissue to the laboratory on ice (Downs et al. 1993; Tang et al. 2012). Arslan et al. (2001) report that in order to

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accurately determine NRA, plant material needs to be moved to the laboratory within a few hours. On the other hand, the same authors emphasize that the lifetime of NR is very short, not to mention the sensitivity of the enzyme to external conditions (Nicholas et al. 1976; Norby et al. 1989). Consequently, the abovementioned methods seem to have many drawbacks. When plants are moved from field conditions to pots in greenhouses, their natural environment and temporary conditions are fully disturbed. On the other hand, transferring segments of leaf tissue on ice to the laboratory interrupts all important environmental and metabolic factors, and the researcher has no control of those changes. Krywult and Bielec (2013) presented a modification of the in vivo NRA assay which allows its use directly under field conditions where plant samples are incubated in a styrofoam box and the temperature of water is adjusted using ice cubes and hot water. This approach has some limitations, especially in highly unstable environments where continuous control of the incubation conditions can be difficult. To meet these requirements, we have invented and constructed a prototype of a portable water bath with a refrigeration option, along with a number of improvements to facilitate research work in the hard-to-access geographic areas and independently of the weather.

Materials and methods Design of the device The housing is white (Figs. 2 and 3) in order to reflect most of the visible radiation, which ensures thermal independence of incubated samples. The entire device is thermally isolated from the external environment by styrofoam and polyethylene foam to prevent rapid heat exchange, which has a crucial effect on battery life. The instrument consists of two modules. The main module (Figs. 1 and 3) includes a stainless steel bowl with water, while a Peltier module (heating/cooling element) is attached to the bottom of the bowl, and a cooling system composed of an aluminum radiator and 12-V fan, electronic driver system based on a microcontroller, and three independent thermometers (two for control of water temperature in the bath and a third for the measurement of outside air temperature). It also features an air humidity sensor. For water temperature homogenization throughout the entire bowl volume, we used a miniature 12-V water pump, which pumps water from the bottom side


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of the bowl (where the heating/cooling element is affixed) and releases it in the upper part of the bath (Fig. 1). This is particularly useful with the use of a refrigeration option because more dense cold water descends from the upper part, equalizing temperature differences. In the power module (Fig. 1), we included a VRLA battery (valveregulated lead-acid battery) of the AGM type (absorbed glass mat) because of its advantages over other types of batteries—it can be mounted in any orientation (Fig. 3) and does not require constant maintenance. The battery current is 12 V and 9 Ah, and these parameters seem to provide the best weight/capacity ratio. The two modules are galvanically connected and can be combined into one integrated device with the attached latches (Fig. 1). The instrument has an input for an AC adapter designed to fit a regular power outlet (when used in a laboratory) and can be also supplied with electricity from a car cigarette lighter via dedicated cables. For an easy transportation of the device, we have installed stable clasps for

shoulder belts (Fig. 3). Due to the considerable weight of the device when it contains water, it is preferable to move the device on one’s back using a dedicated rack (Fig. 3), especially in the case of long-distance walking situations. The rack also has wheels, which are helpful when transporting the device across a flat area (Fig. 3). Inside the bowl of the main module, a tube rack (Figs. 1 and 3) is installed that is suitable for vacuum tubes which are used in the NRA assays (Krywult and Bielec 2013). However, for other types of analyses, different types of racks dedicated to specific glassware shapes can be installed. Both the top cover and battery compartment doors are insulated from water with seals (Fig. 1). Electronic systems are also insulated from environmental factors, allowing field usage even in inclement weather conditions. The device’s (Figs. 1, 2, and 3) weight is about 8.5 kg when filled with water and includes a battery, whereas the weight of the main module alone (with water) is about 5 kg.

Fig. 1 Schematic of the described portable water bath. a Longitudinal section. b Front panel. 21 Main module, 22 power module, 1 lid, 2 seal, 3 snap fastener for belts, 4 carrying handle, 5 tube rack, 6 bowl for incubation water, 7 internal (wet) heat sink, 8 Peltier element, 9 electric water pump, 10 external (dry) heat sink, 11 cooling fan, 12 buckle for stable connection of modules, 13 12-

V battery, 14 insulation material, 15 three-position switch for power selection: battery/AC adapter, 16 three-position switch for water pump regulation: continuous work/intermittent work, 17 screen of main driver for parameter setting and reading, 18 main control buttons, 19 additional, reserve battery thermometer, 20 hatch of battery compartment

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Site selection and experimental species

NRA assay in situ

In order to verify the usefulness of the presented instrument in difficult and highly unstable field conditions, measurement sites were selected in Karkonosze National Park (Poland), at high altitudes (1360–1450 m a.s.l.) across alpine grasslands [Carici (rigidae)–Nardetum and Carici (rigidae)–Festucetum airoids; Żołnierz and Wojtuń 2013] on a plateau above the treeline. The activity of the enzyme of interest was studied in two plant species typical of subalpine ecosystems: one grass, Deschampsia caespitosa, and one dicotyledon, Homogyne alpina. The species were selected randomly at the opportunity to other studies, but there are significant differences in leaf shapes and growth forms of these species which imply different strategies of leaf partitioning for the assay (sterile scissors or hole puncher). Measurements were performed at four sites found at least 3 km from each other, in June and July 2015.

For nitrate reductase activity analysis, we used the in vivo method presented for the first time by Jaworski (1971) and modified by Al Gharbi and Hipkin (1984), Downs et al. (1993), Norby et al. (1989) and Cazetta and Villea (2004) . We followed a protocol developed by Krywult and Bielec (2013) which allows the use of this method in situ. Because of the lack of sufficient electrical power in the investigated area, samples were vacuum infiltrated using a manual vacuum pump with our own modifications. The sampling was carried out between 11:00 and 15:00 GMT only during sunny days, with relatively similar air temperature. The circles of leaves were cut with a hole puncher. Grass leaves were cut into 2–3-mm parts using sterile scissors and immediately placed into test tubes containing an incubation buffer solution and dedicated plugs allowing a connection with a vacuum pump. Samples were vacuum infiltrated at 0.33 atm for 5 min and incubated in a buffer for 2 h at 25 °C in the described portable water bath. The incubation buffer was prepared according to Krywult and Bielec (2013). Immediately after the incubation, 1 ml of the reaction mixture was placed in a test tube and 1 ml of 1% sulfanilamide in 8% HCl and 1 ml of 0.02% N-(1-naphtyl)ethylenediamine dihydrochloride were added. Color produced by the diazotization reaction is stable for 24 h. Hence, we were able to conduct all planned incubations and reactions in the field, transfer samples to a laboratory, and to measure absorbance at 540 nm using a spectrophotometer (model Secomam 250) within 24 h. It should be mentioned that whole analysis could also be performed in the field with the use of a commercially available portable, battery charged pocket spectrophotometer. Leaf samples were removed from the initial test tubes and dried at 60 °C to a constant weight in order to express NR activity as the amount of nitrite synthesized per gram of plant tissue dry mass per hour (μM NO2 g−1 DW h−1). For the NR activity calculation, a standard curve had been prepared earlier.

Fig. 2 Illustrative photo of the described device. On the front panel, the following are seen: main switch for selecting power supply (from AC adapter or battery), control buttons, and LCD screen for temperature programming and displaying various parameters of water inside the bowl and air outside the instrument. Moreover, an additional small thermometer to measure temperature of incubation water and independently powered by a small battery has been installed. This is needed in situations when the main battery is fully discharged or in cases when only the main module is used (separately) in the field

Results and discussion The maximum possible water temperature (at room temperature = 26 °C) that can be obtained in the bowl was 90 °C, and the lowest temperature was 7 °C. It is most efficient to pour water of a desired temperature into the bowl. In such a case, the battery is used only to maintain water temperature and not for the purpose of reaching a


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Fig. 3 Illustration of the described device: a main module and battery module separately, b manner of using belts for carrying the device on a shoulder, c view from the inside (stainless steel water chamber and rack for tubes are visible), d rack for comfortable carrying of the instrument as a backpack, e manner of using the rack, f manner of battery installation in the power module, g bottom of the main module with visible cooling fan

certain temperature in the field. The main battery with its presented properties is able to ensure continuous operation of the device for 1 h and 15 min, but it should be noted that the heating/cooling element of the device is not operating continuously, but with intervals depending on the predetermined temperature hysteresis. The best

hysteresis seems to be −0.5 °C for heating and +0.5 °C for the refrigeration option, which assures proper operation of the device in the field for 4 to 7 h. The presented parameters can be improved by using better quality components and with access to specialized manufacturing workshop. We used an electronic control system that

Fig. 4 Foliar NRA in Homogyne alpina and Deschampsia caespitosa at four different sites located at least 3 km from each other (see text). Error bars are ±1 SE (n = 4). Standard errors for D. caespitosa, 1.8–3.4; for H. alpina, 0.3–0.34

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automatically switches from the heating option to the cooling option depending on the outside temperature, so the researcher does not have to focus on checking the preset parameters all the time. The weather conditions in the summit parts of the Karkonosze mountains were very unstable including cold winds, variable air temperature over short periods of time from low to high, and intermittent rainfall and fog. Despite these difficult weather conditions, the water temperature inside the described water bath was very stable and insensitive to outside air temperature, irrespective of whether the outside temperature was lower or higher than desired. Therefore, the refrigeration option is highly useful and its use in the described device is justified, which is not often required in typical laboratory baths. Using the described water bath, it is possible to perform multiple measurements in various field locations because samples continue to be incubated in stable conditions. All the water protection features installed in the water bath were sufficient for stable and safe operation of its electronic components even during rain events. The obtained results of the NRA for D. caespitosa and H. alpina are shown in Fig. 4. Standard errors and deviations were low, thanks to stable incubation conditions. Our results were highly reproducible considering the nature of the analyzed enzyme and its sensitivity to external factors (Arslan et al. 2001; Kaiser and Huber 2001). Maximum measured NR activity in D. caespitosa was 2.7 μM NO2 g−1 DW h−1 (Fig. 4), while that for H. alpina was much lower: 0.3 μM NO2 g−1 DW h−1 (Fig. 4). Some studies showed similar NRA range for congeneric D. caespitosa species. In Deschampsia flexuosa leaves, the activity of the enzyme strongly depended on the place of growth and soil nitrate amount, ranging from 2.4 μM NO2 h−1 g−1 DW on unfertile places to 3.9 μM NO2 g−1 DW h−1 on nitraterich soils (Trolestra et al. 1995). Other authors showed that Antarctic hair grass Deschampsia antarctica, when grown around penguin rookeries and unprotected by UV radiation, showed maximum NRA of about 0.844 μM NO2 g−1 DW h−1 which is far less than the value of our results for D. caespitosa (Krywult et al. 2013). It can be assumed that due to extremely unfertile soils which D. antarctica is growing on, this species has adapted to low N availability and it is not able to induce higher NRA as D. caespitosa or D. flexuosa even when it grows near more fertile penguin rookeries. In turn, D. caespitosa NRA values obtained in our study are similar to those exhibited by D. flexuosa presented by Trolestra et al. (1995). Subalpine communities above


the treeline in Karkonosze mountains are located on acidic, unfertile soils (Mochnacka and Banaś 2000), thus many plants can be adapted to use ammonium rather than nitrates as a main N source. It can be suspect that relatively low NRA values for H. alpina may be a result of poor adaptation to nitrate utilization by this typical alpine species. It is highly possible that this species preferentially utilizes other nitrogen forms at a higher level. On the other hand, there is growing evidence that anthropogenic nitrogen deposition from air is and will be a growing problem worldwide in the future ( Galloway et al. 2008). This problem is especially true in places such as the Karkonosze mountains due to its location within the center of the so-called Black Triangle, an area recognized as a one of the most damaged by air pollution in Europe. Recent data shows growing amounts of deposited air nitrogen compounds from anthropogenic activity in Karkonosze mountains (Twarowski et al. 2007). In this situation, plants originally adapted to low nitrate N-form availability will receive unnaturally large access to these compounds which can change all nutritional and competitive relations between coexisting species. The presented in situ method allowed for measurements at a few distant locations with samples continuously incubated while samples were collected. The described method also assured that a minimal amount of plant material was used to get reliable NRA results. This is particularly important in cases of research on endangered and protected species. The presented portable and battery-operated water bath allows to incubate samples under well-defined, constant temperature and darkness in situ. The device can yield substantial improvements in ecophysiological studies performed under actual field conditions with reliable results. Acknowledgements The authors are grateful to Dr. Marek Krywult for helpful suggestions and introduction to the NRA in situ method, to Prof. Aleksandra Samecka-Cymerman for critical comments and scientific advice which helped to improve the study, and for Andrzej L. Rudecki for providing valuable technical suggestions in the process of prototype construction.

Author contributions statement A.R. and B.W. conceived the ideas and designed methodology, collected the data, and built the prototype; A.R., B.W., and A.B. wrote the article. All authors contributed critically to the drafts and gave final approval for publication. The presented device has been reported in the Polish Patent Office. Application received unique number: P.417816.


References Al Gharbi, A., & Hipkin, C. R. (1984). Studies on nitrate reductase in British angiosperms. New Phytologist, 97, 629–639. Arslan, H., & Güleryüz, G. (2005). A study on nitrate reductase activity (NRA) of geophytes from Mediterranean environment. Flora, 200, 434–443. Arslan, H., Güleryüz, G., Gökçeoğlu, M., (2001) A comparative study on nitrate reductase activity (NRA) of some endemic plants from Uludağ National Park. Anadolu University Journal of Science and Technology. Calatayud, A., Roca, D., Gorbe, E., & Martinez, P. F. (2007). Light acclimation in rose (Rosa hybrid cv. Grand gala) leaves after pruning: effects on chlorophyll a fluorescence, nitrate reductase, ammonium and carbohydrates. Scienta Horticulturae, 111, 152–159. Cazetta, J. O., & Vasques Villea, L. C. (2004). Nitrate reductase activity in leaves and stems of Tanner grass (Brachiaria radicans Napper). Scienta Agricola, 61, 640–648. Chmura, D., Krywult, M., & Kozak, J. L. (2016). Nitrate reductase activity (NRA) in the invasive alien Fallopia japonica: seasonal variation, differences among habitats types, and comparison with native species. Acta Societatis Botanicorum Poloniae., 85(3), 3514. doi:10.5586/asbp.3514. Dias, T., Neto, D., Martins-Loução, M. A., Sheppard, L., & Cruz, C. (2011). Patterns of nitrate reductase activity vary according to the plant functional group in a Mediterranean maquis. Plant and Soil, 347, 363–376. Diekmann, M., & Falkengren-Grerup, U. (2002). Prediction of species response to atmospheric nitrogen deposition by means of ecological measures and life history traits. Journal of Ecology, 90(1), 108–120 Dovis, V. L., Hippler, F. W. R., Silva, K. L., Riberio, R. V., Machado, E. C., & Mattos Jr., D. (2014). Optimization of the nitrate reductase activity assay for citrus trees. Brazilian Journal of Botany., 37, 383–390. Downs, M. R., Nadelhoffer, K. J., Melillo, J. M., & Aber, J. D. (1993). Foliar and fine root nitrate reductase activity in seedlings of four forest tree species in relation to nitrogen availability. Trees, 7, 233–236. Filippou, P., Bouchagier, P., Skotti, E., & Fotopoulos, P. (2014). Proline and reactive oxygen/nitrogen species metabolism is involved in tolerant response of the invasive plant species Alianthus altissima to drought and salinity. Environmental and Experimental Botany, 97, 1–10. Galloway, J. N., Townsted, A. R., Erisman, J. W., Bekunda, M., Cai, Z., Freney, J.R., Martinelli, L. A., Speitzinger, S. P., & Sutton, M. A. (2008). Transformation of the nitrogen cycle: Recent trends, questions, and potential solutions. Science, 320(5878), 889–892 Harper, J. E., & Hageman, R. H. (1972). Canopy and seasonal profiles of nitrate reductase activity in soybeans (Glycine max L. Merr.) Plant Physiology, 49, 146–154. Jaworski, E. G. (1971). Nitrate reductase activity in intact plant tissues. Biochemical and Biophysical Research Communications, 43, 1274–1279. Kaiser, W. M., & Huber, S. C. (2001). Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers. Journal of Experimental Botany., 52, 1981–1989.

Page 7 of 7 332 Krywult, M., & Bielec, D. (2013). Method of measurement of nitrate reductase activity in field conditions. Journal of Ecological Engineering., 14, 7–11. doi:10.5604/2081139 X.1031524. Krywult, M., & Bytnerowicz, A. (1997). Induction of nitrate reductase activity by nitric acid vapor in California black oak (Quercus kelloggii), canyon live oak (Quercus chrysolepis), and ponderosa pine (Pinus ponderosa) seedlings. Canadian Journal of Forest Research., 27(12), 2101–2104. Krywult, M., Turnen, M., Sutinen, M. L., Derome, K., & Norokorpi, Y. (2002). Nitrate reductase activity in some subarctic species and UV influence in the foliage of Betula pendula Roth. seedlings. Science of the Total Environment., 284, 149–155. Krywult, M., Smykla, J., & Wincenciak, A. (2013). The presence of nitrates and the impact of ultraviolet radiation as factors that determines nitrate reductase activity and nitrogen concentration in Deschampsia antarctica Desv. around penguin rookeries on King George Island, maritime Antarctica. Water Air Soil Pollution, 224, 1563. McGill, B. J., Enquist, B. J., Weiher, E., & Westoby, M. (2006). Rebuilding community ecology from functional traits. Trends in Ecology and Evolution, 21, 178–185. Mochnacka, K., & Banaś, M. (2000). Occurrence and genetic relationships of uranium and thorium mineralization in the Karkonosze-Izera block (the Sudety MTS, SW Poland). Annales Societatis Geologorum Poloniae, 70, 137–150. Nicholas, J. C., Harper, J. E., & Hageman, R. H. (1976). Nitrate reductase activity in soybeans (Glycine max [L.] Merr.) Plant Physiology, 58, 731–735. Norby, R. J. (1989). Foliar reductase: a marker for assimilation of atmospheric nitrogen oxides. In G. M. Woodwell (Ed.), Biologic markers of air pollution stress and damage in forests (pp. 245–250). Washington D.C: National Academy Press. Norby, R. J., Weerasurija, Y., & Hanson, P. J. (1989). Induction of nitrate reductase activity in red spruce needles by NO2 and HNO3 vapor. Canadian Journal of forest research., 19, 889– 896. Sakar, F. S., Arslan, H., Kirmizi, S., & Güleryüz, G. (2010). Nitrate reductase activity (NRA) in Asphodelus aestivus Brot. (Liliaceae): distribution among organs, seasonal variation and differences among populations. Flora, 205, 527– 531. Tang, M. H., Porder, S., & Lovett, G. M. (2012). Species differences in nitrate reductase activity are unaffected by nitrogen enrichment in northeastern forests. Forest Ecology and management., 275, 52–59. Trolestra, S. R., Wagenaar, R., Smant, W., & De Boer, W. (1995). Soil nitrogen transformations and nitrate utilization by Deschampsia flexuosa (L.) Trin. at two contrasting heathland sites. Plant and Soil, 176, 81–93. Twarowski, R., Gendola, T., Liana, E., Wostek-Zgraba, K. (2007) Deposition of pollutants from atmosphere with precipitation in The Giant Mountains in the years 1994–2004. In: Štursa, J., Knapik, R. (eds), Geoekologické problémy Krkonoš. Sborn. Mez. Věd. Konf., říjen 2006, Svoboda n. Ŭpou. Opera Corcontica, 44/1, 213–225. Żołnierz, L., Wojtuń, B. (2013). Roślinność subalpejska i alpejska. In R. Knapik & A. Raj (Eds.), Przyroda Karkonoskiego Parku Narodowego (pp. 241–278). Jelenia Góra: Karkonoski Park Narodowy 241–278.