ECOPHYSIOLOGY OF KEY ORGANISMS IN THE ECOSYSTEM

Offshore Inshore

3. ECOPHYSIOLOGY OF KEY ORGANISMS IN THE ECOSYSTEM

Different flavonoid patterns in Deschampsia antarctica and Colobanthus quitensis from the marine Antarctic

Cornelius Lütz*, Maria Blassnigg, Daniel Remias

Dept. Physiology and Cell Physiology of Alpine Plants, Institute of Botany, University of Innsbruck, Innsbruck, Austria, cornelius.luetz@uibk.ac.at; *corresponding author

Introduction

Life in Antarctica is often seen as being established in an extreme environment.

However, evolution has shown that organisms were able to adapt to this envi-ronment. In some cases they retained their climate adaptation raised in other environments and thus managed to grow for a long time in Antarctic regions, like both higher plants spread over the Antarctic Peninsula, Deschampsia ant-arctica and Colobanthus quitensis. The original natural occurrence is the high altitude region of the Andean mountains. Their distribution over the Antarctic Peninsula is well documented (cf. Moore 1970, Komárková et al. 1990). The ecophysiological survey given by Alberdi et al. (2002) showed, that physiologi-cal studies of both plants are scarce, especially if selected for data raised in the Antarctic environment. Edwards and Smith (1988) measured photosynthesis and respiration of plants after transfer to a lab in England. Experiments on the physiological status of the plants with some measurements performed on Robert Island (not far from King George Island), but mostly under lab condi-tions, come from Zúñiga et al. (1994, 1996) or Perez-Torres et al. (2006). Stud-ies on cold resistance in Antarctic angiosperms also address anti-freeze pro-teins (Bravo et al. 2001, Olave-Concha et al. 2005). A field study on CO2 -fixa-tion and Chl fluorescence of C. quitensis and D. antarctica was published by Xiong et al. (1999).

Many reports on increased UV-irradiation over Antarctica or the Arctic have been published (Hempel 1994, Björn et al. 1999, Huiskes et al. 2003).

Marine ecosystems seem to be disturbed by such ozone-hole activities, especially during the Austral spring from October to December, when sea ice cover melts away and UV intensities reach their maximal values (Wiencke et al.

1998, Kirchhoff and Echer 2001, Beyer and Bölter 2002). But a clear conclusion whether terrestrial life is endangered by increased UV-irradiation has not been drawn so far.

Only few papers deal with possible influences of UV-B on both higher plants from Antarctica. Day et al. (1999) performed a UV-B reduction and exclu-sion experiment in the field and measured growth, bulk plastid pigments and flavonoids. However, UV-B reduction experiments will not allow predictions on plant metabolism under an increase in UV-B. Ruhland and Day (2001) extended these studies to describe influences of reduced versus ambient UV-B on seed banks and total pigments in C. quitensis.

Flavonoids are the main shielding compounds against UV-irradiation, but

The comparison may show whether quantitative or qualitative changes took place with growth time; to our knowledge these analyses are the first direct comparison of flavonoid composition between both plants.

Materials and Methods

During two research expeditions to King George Island (K.G.I., maritime Ant-arctic, 62°20’S) in 2003 and 2006, samples from D. antarctica and C. quitensis were collected on the open shores of the island in walking distance from the Dallmann research station. At the selected sampling sites, the plants were abundantly growing. Leaves of 100-150 mg weight were cut from the plants and transferred into vials containing 5 ml methanol per sample. On average, 5 sam-ples per species per sampling date were prepared. Sampling periods were January and February, 2003 and 2006, mostly between 10 am and 4 pm. Addi-tional samplings were done after some plants having been transferred to the Institute of Botany in Innsbruck, Austria. They were kept growing in an unheated glass-house under natural light but only obtaining approx. 10% of the ambient UV. Growth temperature for these plants exceeded average outside tempera-ture by about 3-5 °C.

Climate conditions: during January and February at King George Island in 2003:

mean daily temperatures: +2 to +9°C, in wind protected sample plots up to +14°C (in air close to cushion), often sunny, two days with light snow fall, occa-sionally overcast, seldom fog. Observations for 2006: mean daily temperatures of 0 to +7°C, occasionally sunny (31.1.06), often overcast, windy, several snow falls (19.2.06). Own simple PAR light measurements showed 500 –900 µMol photons on overcast days, but up to 2300 µMol photons PAR on sun exposed cushions in direction of the sun, as often leaf orientation is. Detailed meteoro-logical data, over longer periods of time, accompanied by landscape and geo-logical information, can be found in Wiencke et al. (1998) and Beyer and Bölter (2002).

Flavonoid assays: The samples (leaves in methanol) were stored until transport back to the Institute of Botany at –20°C; later until processing at –53°C. During storage about all flavonoids were extracted into the solvent, therefore two small volumes of methanol for re-extraction removed remaining pigments completely.

Separation and quantification was done on an Agilent-HPLC system with diode array absorbance spectra detection according to the method described by Turunen et al. (1999), which also was found to be useful to describe flavonoids in a number of alpine and arctic plants. Semi-quantification of peak amounts were performed as relative absorption units per g fresh weight. In both plants, the special leaf structure did not allow an accurate measurement of leaf surface as an additional reference.

Results and Discussion

The experimental setup allows a good extraction and separation of soluble fla-vonoids, but the tightly cell wall bound compounds, including most phenylpro-panes, could not be assayed. The separation of the vacuolar flavonoids by means of high resolution HPLC with samples harvested in 2003 resp. 2006 showed nearly identical composition in both species, no additional peaks were found in this comparison. In Fig. 1 representative separations are given. Six

main compounds with absorptions in the spectral region of 300-400 nm were selected as possible UV screening pigments for quantification and further iden-tification. Other, always minor peaks recognized at the detection wavelength of 280 nm, but without relevant absorption above 290 nm (lower limit of current UV-B/UV-A irradiation input) were not considered. In C. quitensis, these com-pounds appeared between 25.0 and 31.0 min separation time with B, C and E as main peaks. In D. antarctica, the selected peaks separated between 18.0 and 31.5 min, main compounds are C and D. Both separations indicate a quite different, plant specific composition of UV-A absorbing flavonoids.

The insets in Fig. 1 present absorption spectra of different peaks: in C.

quitensis: only peak A absorbs maximally at 313.5 nm, peak D at 338.0 nm and peaks B, C, E and F at 349.0 nm. The data for D. antarctica: peaks A and B:

324.5 nm, C: 350.0 nm, D,E and F: 352.0-353.0 nm. In table 1 the relative amounts of flavonoids are given for three samplings in 2003, two samplings in 2006, and for some samples taken from plants which, after the transfer from Antarctica to the Institute of Botany, were kept in a greenhouse (see Methods).

For samples extracted directly at King George Island, C. quitensis showed relative constant amounts of peaks A to E over the sampling period in 2003, only peak F is reduced by about 30%. In 2006 (the first harvest occurred 10 days later compared to 2003) during the 20 days span between samplings most compounds were strongly reduced. Except peak A, the plants from 2003 accumulated more flavonoids than those in 2006. Already from visual observa-tion, C. quitensis seemed to develop senescence in February, which may explain the reductions especially for peaks B, C, E and F. Interestingly, the amounts measured at 31.1.2006, before senescence started, represent about the amounts in average from 2003.

Despite the different weather in both years (see methods), there is a remarkable stable equipment of UV-A absorbing flavonoids in this plant. After transfer of such plants to the institute for growth in a greenhouse under warmer and reduced UV conditions, no new compounds could be detected via HPLC.

Strong reductions occurred in all peaks except for F, which remained about constant in concentration.

For D. antarctica, harvested on the island in 2003, a continuous reduction of all compounds with growth time occurred. In 2006, the 20 days of difference between the two harvests showed reductions only for peaks C and D, while B and F increased strongly, and peak A and E did not change considerably. Gen-erally, this plant accumulated much more compounds in 2006. D. antarctica as a typical Poaceae seems to be more stable in growth because of continuous development of leaves from their basis compared to C. quitensis as a Caryo-phyllaceae. The former adopts to colder weather in February with increasing some flavonoids, as it is well known for several other species, via induction of the phenylpropane metabolism (Grace 2005), while the latter starts to decom-pose some constituents during the Antarctic autumn. Growth of D. antarctica under greenhouse conditions again resulted in strong reduction of all com-pounds, and in parallel to C. quitensis, compound F keeps about the same level after 6 months in the 2003 samples, and is reduced further 2.5 years later. The 6 months glasshouse period for the 2006 samples support the expected

reduc-Fig.1. Separation of flavonoids and related compounds from C. quitensis (upper) and D. antarctica (lower). Peak labelling indicates the compounds as are listed in tab. 1. Detection wavelength: 280 nm; the labelled peaks have additional absorption maxima between 300 and 400 nm (insets).

10 15 20 25 30 35

Colobanthus quitensis