1 Introduction

The majority of poikilohydric vascular plants (“resurrection plants”) occur in arid and semi-arid regions of southern and southwestern Africa, southern America and Western Australia (Gaff 1977, 1987). They predominantly colonise shallow rocky soils in their sub-tropical habitats, often on inselbergs (Porembski and Barthlott 2000). Desiccation-tolerant angiosperms are found within both monocotyledons (Cyperaceae, Poaceae and Velloziaceae) and dicotyledons (predominantly the ex-Scrophulariaceae). These poikilohydric cormophytes are exposed to severe drought during long dry seasons (5–10 months per year). Hartung et al. (1998) pointed out that the mechanisms for drought tolerance from the molecular to the physiological and anatomical level are rather costly and with the consequence of a selective disadvantage under most less extreme growing conditions.

Among the Scrophulariaceae Chamaegigas intrepidus Dinter, formerly Linderniaintrepidus (Dinter) Oberm. (Fig. 12.1), is rather unique. This aquatic resurrection plant occurs in shallow, only temporarily water-filled rock pools on granite outcrops in Namibia (Giess 1969). Hundred years ago this plant was discovered in 1909 by the German botanist Kurt Dinter 12 km east of Okahandja in Central Namibia. He had mentioned this plant already in 1909 (Dinter 1909). He gave the plant the scientific name, which means in German “Unerschrockener Zwergriese” (“undaunted dwarf giant”), since he was very much impressed by the plant surviving the extreme conditions at its natural habitat (Dinter 1918). High temperature and high evaporative demand caused the bottom of the pool in which the plant grew to remain permanently dry for at least half a year. The plants survived as tiny rhizomes (diameter about 1 mm) and shrivelled leaves densely covering the bottom of the pool in a 1 cm thick layer of sand grains, dehydrated algae, dead daphnias, animal faeces and leaf litter. In spite of the high solar irradiation, extreme temperatures and nearly completely dry air, a dense mat of small green Chamaegigas leaves covered the bottom of the pools within minutes after the first summer rainfall had filled the small pools. Two days after that Dinter could see pink-coloured flowers in the midst of small rosette leaves floating on the water on top of a thin stem.

Fig. 12.1
figure 1_12

Flowering rosette of Chamaegigas intrepidus with lanceolate submerged leaves (8–15 mm long; open arrow), inserting on short rhizomes, and two pairs of decussate floating leaves (filled arrow) on a 10 cm long stem with two leaves. For a more detailed description of the plant, especially the flowers, refer to Fischer (1992) and Woitke et al. (2006)

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Chamaegigas is adapted to the fluctuations of wet and dry conditions at its habitat through its ability for fast de- and rehydration. When water from the pools has been lost through evaporation, plants dry within less than 2 h (Gaff and Giess 1986). After rewatering, the vegetative organs regain full metabolic activity within 2 h (Hickel 1967). Rosettes kept for 4 years desiccated could be revived successfully including the formation of flowers (O.H. Volk, personal communication); after 6 years, however, rosettes only became green. Floating leaves and flower buds were not formed (own observation). The extremely fast recovery after rehydration and the long survival in the desiccated state makes Chamaegigas by far the most impressive of all poikilohydric angiosperms. In the following, habitat conditions and the plant’s mechanisms of adaptation to its complex stressful environmental conditions at the anatomical, biochemical and physiological level are described as well as implications of its isolated geographical distribution for generative reproduction and gene flow.

2 Distribution and Habitat

Chamaegigas grows endemically in Namibia (Fischer 1992), exclusively in areas with granite outcrops (inselbergs) in the semi-desert and savanna transition zone (Giess 1969, 1997). The habitats of the species are in the arid to semi-arid region with 160–570 mm precipitation per year, with rainfall on only 20–70 days during summer (November to April), and a high variability from year to year. During the wet season, a few (5–12) rainy days alternate with a number (up to 60) of dry days (Hickel 1967). The shallow rock pools (maximum water level ca. 15 cm) usually dry out completely during dry days. Over the whole wet season, these ephemeral pools may be filled with water for some 40–85 days in total. Thus, the plant may experience 15–20 rehydration–dehydration cycles during a single rainy season (Gaff and Giess 1986). Annual average temperature is 20°C. During the dry season, air temperatures may rise up to 42°C, and sun-exposed rocks heat up to 50°C at least (Dinter 1918). Average air humidity is 40% and at the end of the dry season (September) 22% only (Hickel 1967).

Due to the thin layer of debris at the bottom of the rock pools, the water is very poor in nutrients. Furthermore, after extensive rainfall the shallow pools may overflow, thus leaching mineral nutrients from the sediment (Gaff and Giess 1986). This can cause more severe nutrient deficiencies, especially when there is plenty of water for sustained growth. On the other hand, wild and domestic animals deposit urine and dung on the rock surface, which are subsequently washed into the pools by rain water (Heil 1924). Thus, especially during the early part of the wet season, there may be high amounts of urea and perhaps other dissolved organic nitrogen (DON) compounds present in the water, which are steadily diluted by leaching and plant uptake during the growth period.

Chamaegigas is physiologically active during the warm rainy season, whereas it survives the long (up to 11 months) dry season in the dehydrated state (Hickel 1967). Apart from the intense solar irradiation, high air temperature and low air humidity during the dry season, the plants are exposed to recurrent flooding and drought, low nutrient contents and drastic diurnal fluctuations of pH of the water during the wet season. Thus, this species suffers from a complex set of extremely harsh and interacting environmental conditions.

3 Site Description

Most of the data described here originate predominantly from field experiments performed at one of the species’ natural growing sites on the farm Otjua (Omaruru District, Namibia, 21°10′S, 16°E). The farm lies in the thornbush savanna at an elevation of about 1,400 m above sea level. Vegetation is dominated by Acacia spp. In between the vegetated area, granite outcrops rise to a height of about 20 m with a length of several hundred metres. Due to high temperature fluctuations between day and night, thin sections of granite burst from the rock surface. Subsequent erosion by wind deepens the shallow holes to a maximum depth of about 0.3 m. During the wet season, these small depressions become filled with rain and run-off water from the outcrops and partly from overflow from adjacent pools.

In pools with a water level of more than 15 cm (with the homoiohydric aquatic plant Limosella grandiflora and/or C. intrepidus), there was a high abundance of tadpoles. Pools with a high nutrient input from dung were heavily populated by algae of the genus Spirogyra. At the foot and in cracks of the outcrops, the terrestrial resurrection plants Craterostigma plantagineum Footnote 1, Xerophyta humilis and Eragrostis nindensis, the poikilohydric liverwort Exormotheca bulbigena (Marchantiales) and the aufi Riccia species R. angolesis, atropurpurea, crinita, nigrella, okahandiana, rosea and runssoriensis occurred (Bornefeld and Volk 2002).

4 Environmental Stress Conditions

4.1 Air Temperature and Humidity at the Rock Surface

When pools are not filled with water, the dry Chamaegigas plants are exposed to temperatures up to 60°C, which is approximately 20–30°C higher than water temperature when the plants are physiologically active. Diurnal amplitudes of air temperature between day and night may exceed 30°C, due to large losses of energy during cloudless nights.

During days without rainfall, minimum air humidity decreases to 5–10%. Maximum air humidity at night (ca. 40%) does not indicate dewfall. As a consequence of the high evaporative demand of the air, actual evaporation exceeds 10 mm day−1. On more cloudy days, average minimum humidity is 10–20%, with 70 to nearly 100% at night.

4.2 Water Level and Conductivity

Water level in water-filled pools with Chamaegigas ranged from 4 to 13 cm (median 7.5 cm). In pools drying out loss of water caused the dissolved compounds to be concentrated as measured by the electrical conductivity. A decrease in water level by a factor of two increased conductivity on average up to twofold (Fig. 12.2a) and in some cases up to fourfold. After refilling by rain, conductivity decreased to rather low values (<50 μS cm−1). This indicates that towards the end of the wet season, only minute amounts of nutrients are washed into the pools from the surrounding rock surface.

Fig. 12.2
figure 2_12

Temporal course of (a) water level and electric conductivity, (b) water temperature and pH, (c) ammonium and urea concentration during desiccation in a temporarily water-filled rock pool on a granite outcrop on the farm Otjua (Omaruru District, Namibia). Floating leaves of Chamaegigas intrepidus covered the pool surface by ca. 25%. Filled bars indicate nighttime hours, open bars daytime hours (modified after Schiller et al. 1997; Heilmeier and Hartung 2001)

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4.3 Temperature and pH of the Pool Water

Water temperature showed similar diurnal oscillations as air temperature, however, with a smaller amplitude. Maximum values reached ca. 35°C during the day and minimum temperatures at night were comparable to nocturnal air temperature (Fig. 12.2b). Parallel to diurnal fluctuations of water temperature were oscillations in pH of the pool water, with maximum values at afternoon and minimum values during night. While minimum pH, similarly to minimum water temperature, stayed constant for several nights, maximum pH values varied in accordance with maximum water temperature Table 12.1. Thus, during hotter days, diurnal amplitudes in pH were larger than during cooler days. Maximum pH values recorded in flooded pools in 1998 were near 10 in the afternoon and minimum near 5 in the morning. Still higher pH values in the afternoon (pH 12) were measured in 1995 (Schiller et al. 1997).

Table 12.1 Amplitudes of temperature and pH in different temporarily water-filled rock pools on granite outcrops on the farm Otjua (Omaruru District, Namibia), inhabiting the aquatic resurrection plant Chamaegigas intrepidus (T min, minimum temperature; T max, maximum temperature; pHmin, minimum pH; pHmax, maximum pH; n.d., no data)
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4.4 CO2 and HCO −3 Concentration of the Pool Water

Diurnal oscillations of pH were mainly a consequence of changing solubility of CO2 due to fluctuations of water temperature. During the daytime CO2 was completely lost from the pools by diffusion when water temperature was above 30°C. This raised the pH to strong alkaline values up to pH 10. Most of the inorganic carbon was present in the form of HCO −3 , with peak concentrations about 100 times higher than maximum CO2 concentrations during the night. After sunset, when water temperature steadily decreased, CO2 concentration increased, initially with a high rate, throughout the whole night due to respiration. This caused a rapid decrease in pH down to slightly acidic values. However, as soon as water temperature increased in the morning, CO2 concentration rapidly dropped to zero. Thus, for submerged Chamaegigas plants CO2 is not available for photosynthesis during most part of the daytime.

4.5 Concentration of Mineral Nutrients in the Pool Water and the Sediment

The most abundant metallic cation in the water of pools with Chamaegigas was sodium (average concentration 190 mmol m−3), followed by calcium (70 mmol m−3), potassium and magnesium (30 mmol m−3 each). Median concentrations of chloride and sulphate were about half that of Na+ and Ca2+, respectively. Phosphate concentrations were below detection limits in most of the cases.

Mineral nutrient contents in the sediment of the rock pools at the end of the dry season are in most cases higher than those given by Gaff and Giess (1986) for Chamaegigas rock pools. This is especially true for potassium, magnesium (ca. 5 g kg−1 dw each) and phosphorus (ca. 0.6 g kg−1 dw), whereas the content of total nitrogen (3.5 g kg−1 dw) is somewhat lower than the value from Gaff and Giess (1986).

Among nitrogenous compounds, both inorganic and organic N species were considered as possible nitrogen sources for Chamaegigas. In contrast to temporary rock pools in the Namib-Naukluft Park (Kok and Grobbelaar 1985), nitrate was below detection limit in most of the cases. Ammonium, however, was much more abundant, with maximum concentrations in drying pools of 0.6 mM (Fig. 12.2c). Amino acids were also present in the low micromolar concentration range (Schiller et al. 1998b). In flooded pools, the most abundant amino acids were glycine, serine and asparagine. Less common, although also with high concentrations when present, were glutamine and alanine. During drying, the relative proportions of amino acids changed, with asparagine, serine and glycine found in nearly all samples. Four amino acids could be detected in almost dry pools only, namely glutamine, threonine, arginine and tyrosine.

The most abundant N compound in both flooded and drying pools was urea. The general time course of urea concentration during drying out of the pools was similar to NH +4 (Fig. 12.2c). Evaporation of the water caused a dramatic increase for both compounds, which was, however, more pronounced in NH +4 .

5 Anatomical Features of C. intrepidus

Fully hydrated leaves of Chamaegigas do not exhibit xeromorphic anatomical features. Their submerged leaves show, however, a remarkable shrinkage during desiccation. The length of the desiccated leaves is 10–20% of the hydrated leaves (Heil 1924; Hickel 1967; Schiller et al. 1999). Thereby, they withdraw to the sediment where they are protected against the extremely high light intensities. This drastic shrinkage has been attributed to the unique “contractive tracheids”. These xylem elements that exist in the submerged leaves only [although Hickel (1967) reports that a few may exist in floating leaves also] have thin longitudinal cell walls with broad spiral strengthenings and terminal plates with a small lumen. The shrinking process is mainly due to these specialised xylem elements that contract like an accordion. Maximum shrinkage is limited to living leaves. The floating mesophytic, homoiohydric leaves that exhibit a normal bifacial anatomy shrink by only 15–20% during desiccation, as it is usually the case for mesophytic plants.

Only little information has been published about the root anatomy of Chamaegigas. Heil (1924) and Hickel (1967) described the rhizodermis, which is absolutely free of root hairs, and the dimorphic exodermis with short cells. Heil (1924) observed a distinct aerenchyma with radial rows of tiny cells in the cortex. Different from submerged leaves, a minute longitudinal shrinkage of Chamaegigas roots by 3% only was observed with our plant material (Heilmeier et al. 2002). In contrast to Hickel (1967), however, lateral shrinkage between 30 and 35% was recorded, which is the result of an exclusive shrinkage of the rhizodermis and the exodermis. This conclusion is supported by scanning electron microscopic investigations of desiccated Chamaegigas roots (Heilmeier et al. 2002). Contrarily to Heil (1924), no aerenchyma could be detected. The root cortex consists of a single layer of extremely large cortical cells with extremely thin cell walls and an endodermis (Fig. 12.3). The central stele exhibits a small and simple diarchic xylem. Despite the extreme fragile appearance of the large cortical cells, no shrinkage or collapse could be observed after complete desiccation. The thin walls of the large cortex cells must be extremely stiff to resist shrinkage. No remarkable staining that could point to an extraordinarily chemical composition of cell walls could be seen as it seems to be the case with the cell walls of leaves of Craterostigma wilmsii that contain both xyloglucans and unesterified pectins particularly in dehydrated cells (Vicrè et al. 1999). As both these compounds are known to strengthen cell walls, it would be interesting to examine whether cortical cell walls of Chamaegigas roots also exhibit a similar chemical composition. The unique stability of the large cortical cell walls seems to be an additional part of the adaptation of the plant to its extreme environment as it minimises shrinking of the roots during desiccation and thus maintains their physical structure within the sediment. Intracellular spaces are small and vacuum infiltration of hydrated roots with water resulted in a fresh weight increase of approximately 10–15% only (Heilmeier et al. 2002). This also supports the conclusion that the cortex of Chamaegigas is not formed by an aerenchyma.

Fig. 12.3
figure 3_12

Scanning electron micrograph of a cross section from a desiccated root with large cortex cells. The shrunken rhizodermis and hypodermis form a velamen-type layer

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As demonstrated already by Heil (1924) and Hickel (1967), the root exodermis contains passage cells with outer cell wall thickenings which are known from aerial roots of orchids and of some roots of plants within the family of Asclepiadaceae (Guttenberg 1968). They are believed to be an adaptation of roots to drought and are not known for roots of homoiohydric aquatic plants. Hickel (1967) concludes that in desiccated roots, when the collapsed rhizodermis and exodermis form a layer that resembles a velamen radicum, the outer pad, which would close the passage cells, acts like a valve and slows down the water loss.

6 Physiological, Biochemical and Molecular Adaptations to Stress in C. intrepidus

6.1 Intracellular pH Stability

Chamaegigas has to cope with substantial diurnal fluctuations in the pool water pH. 31P NMR spectroscopy has been used to investigate the effect of external pH and dehydration on intracellular pH of Chamaegigas roots and leaves (Schiller et al. 1998a). High external pH (10.0) caused only a negligible alkalisation of the root cytoplasm, and drastic dehydration caused a small alkalisation of leaf vacuoles at pH 10. These results imply an unusually effective pH regulation consistent with the adaptation of Chamaegigas to a large number of adverse environmental factors. The NMR analysis also showed that dehydration had no effect on the pools of inorganic phosphate and phosphocholine. This indicated that membranes are most effectively protected from damages due to low water potentials, because membrane damage is usually accompanied by an increase of inorganic phosphate and phosphocholines.

6.2 Photosynthesis

Heilmeier and Hartung (2001) determined oxygen and bicarbonate concentration, pH and temperature of a pool covered by 25% with Chamaegigas and observed O2 production even under conditions when the pool water was nearly free of CO2. They concluded that HCO –3 is a carbon source for submerged Chamaegigas leaves. Floating leaves can take up CO2 via stomata on the upper side of the leaves, which seem to be locked open because most of the stomata stay open all the time, even in darkness. This has been observed for many other floating photosynthesising systems like fronds of Lemna (Landolt and Kandeler 1987).

Woitke et al. (2004) concluded from measurement of chlorophyll fluorescence that 75% of total plant photosynthesis takes place in floating leaves. Using this technique, they also detected some desiccation tolerance of floating leaves within 1–2 days. Whether such tolerance exists also over longer periods, as it is the case for submerged leaves, remains very doubtful.

6.3 Nitrogen Nutrition

As shown above, Chamaegigas has to cope with extreme conditions of nitrogen deficiency. Nitrate is virtually absent in the rock pools. Urea, ammonium ions and the amino acids glycine and serine occur in low concentrations, in the micromolar range. Urea cannot be utilised by Chamaegigas directly. Heilmeier et al. (2000) performed uptake experiments with 14C- and 15N-labelled urea. No incorporation and utilisation of urea could be observed, even after long incubation periods (up to 5 days), when the root systems were cleaned carefully. When the natural sediment or jack bean urease was present in the medium, urea-N was accumulated in tissues of Chamaegigas. Furthermore, 15N NMR spectra performed with Chamaegigas roots after incubation with 15N urea did not show any 15N signals such as ammonium, glutamine and glutamate in the absence of urease. However, when ammonium was released by the action of urease 15N could be utilised by Chamaegigas. Urease, therefore, plays an essential role for the acquisition of urea-N. It is important to note in this context that urease can survive the harsh conditions in the desiccated rock pools during the Namibian winter at temperature up to 60°C, complete dryness and high UV irradiation. This remarkable resistant enzyme seems to be a key factor for survival of Chamaegigas (and also of other plants that depend on urea-N deposited from animals).

Ammonium, which originates from urea as shown above especially in drying pools (cf. Fig. 12.2c), may become extremely deficient, especially in highly alkaline pool water where it can be lost as ammonia to the atmosphere. Then Chamaegigas switches to utilisation of glycine, which is most abundant among amino acids in the pool water, as shown after incubation with 15N glycine, both under laboratory and field conditions (Schiller et al. 1998b). 14C glycine can be taken up by Chamaegigas roots with a high-affinity glycine uptake system (K M = 16 μM). Since uptake of glycine is strongly reduced under alkaline conditions (Schiller et al. 1998b), nitrogen utilisation must be expected to occur mainly during the morning hours only.

When Chamaegigas rosettes or isolated root systems were incubated with 15N glycine, 15N NMR spectra showed a glycine, serine, glutamine and glutamate signal (Hartung and Ratcliffe 2002). This agrees with the action of glycine decarboxylase (GDC) and mitochondrial serine hydroxy methyl transferase (SHMT; C1-metabolism, Mouillon et al. 1999) in the Chamaegigas roots. In this case, GDC would release CO2 and ammonium from glycine, and NH +4 would be incorporated into glutamine and glutamate. The second product of glycine metabolism, methylenetetrahydrofolate (M-THF) would then transfer one carbon to another glycine molecule to form serine. Roots are believed to have extremely low (Walton and Woodhouse 1986) or even to lack GDC (Bourguignon et al. 1993), although 15N NMR spectra of 15N glycine supplied maize root tips showed signals similar to those of Chamaegigas roots. The latter, however, seem to metabolise glycine much more rapidly than maize roots (Hartung and Ratcliffe 2002). This could be a part of an adaptation to the natural habitat with glycine as one of the dominating N sources.

Serine, the other dominating amino acid of the rock pools, is taken up by Chamaegigas roots with similar rates as glycine (Schiller et al. 1998b). Assuming that roots of Chamaegigas exhibit activity of SHMT as shown for sycamore suspension cells by Mouillon et al. (1999), glycine again would be a product of serine metabolism and it could be metabolised as shown above. Indeed, different from Zea mays, 15N NMR spectra of Chamaegigas roots that have been pre-incubated with 15N serine exhibited a glutamine and a glutamate signal (Hartung and Ratcliffe 2002).

Glycine has been shown earlier to be the dominant N source for arctic and alpine plants (Chapin et al. 1993; Raab et al. 1996) and a number of sedges from different ecosystems (Raab et al. 1999). Schmidt and Stewart (1999) have demonstrated glycine as the main soil-derived N source for a wide range of wild plants in Australian communities. Chamaegigas is the first example of an aquatic vascular plant showing that amino acids such as glycine and serine can be an important N source.

6.4 Abscisic Acid

The role of abscisic acid (ABA) as a stress hormone seems to be well established. Its biosynthesis is increased especially under drought stress conditions. After transport to the target cells ABA mediates responses that are major components of plant survival mechanisms under stress conditions. Of particular importance is the ABA-controlled stomatal movement, which, however, should be of minor importance in Chamaegigas. The leaves are either submerged or floating on the water surface. Stomatal movement cannot be expected under those conditions. Additionally stomata of water plants very often are non-functional. The anatomy of Lemna guard cells does not allow movement; they are “locked open” (Landolt and Kandeler 1987). Both Heil (1924) and Hickel (1967) investigated stomata of Chamaegigas, however, without any conclusions concerning their function. Epidermal preparations of Chamaegigas floating leaves, published by Heil (1924), only show open stomata. Own unpublished scanning electron microscopical studies of the surface of floating leaves also show open stomata only.

6.4.1 ABA Content Under Control Conditions and Drought Stress

The ABA content of leaves of the poikilohydric angiosperms C. plantagineum, Myrothamnus flabellifolia and Borya nitida is very high under both hydrated and desiccated conditions (0.3–3 nmol g−1 dw; Schiller 1998). Drought-dependent fluctuations, however, are very small. Desiccation causes not more than a two- to threefold increase. In contrast, the ABA concentration in hydrated organs of Chamaegigas is much lower (0.05–0.25 nmol g−1 dw), similar to what is found in a wide range of mesophytic plants. During desiccation, however, a 20- to 30-fold increase in submerged leaves and roots can be observed. The desiccation-dependent ABA increase in the homoiohydric floating leaves, however, is never more than fivefold.

The ABA content of both roots and submerged leaves is well related to the tissue osmotic potential (Fig. 12.4). Different from many mesophytic species no threshold of osmotic potential has to be reached in Chamaegigas to trigger ABA accumulation. It seems that ABA biosynthesis responds more sensitively to water shortage in Chamaegigas than in other plants (Schiller et al. 1997).

Fig. 12.4
figure 4_12

The relationship between osmotic potential and tissue ABA concentration in roots and submerged leaves of Chamaegigas intrepidus (modified after Schiller et al. 1997)

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6.4.2 Oxidative Degradation of ABA

When Chamaegigas tissues were pre-incubated with 14C-ABA, phaseic acid (PA), dihydrophaseic acid (DPA) and conjugates (predominantly glucose esters) of ABA, PA and DPA could be extracted – the same ABA metabolites as in any other plant (Schiller 1998). The response of Chamaegigas to the inhibitor of oxidative ABA degradation, tetcyclacis, is also similar to reports from the literature. Degradation, however, is much slower in Chamaegigas tissues than in those of mesophytic plants such as Valerianella locusta. After 24 h pre-incubation with labelled ABA 3% of the label were recovered in PA in Chamaegigas, but 31% in V. locusta. Leaves of many other plants metabolise ABA even more rapidly (90–100% within 1 day). The slow degradation rates of ABA in Chamaegigas contribute to maintain high ABA concentrations, similarly as it happens when PA formation is inhibited by substances such as tetcyclacis or paclobutrazol.

6.4.3 ABA Conjugates

In Chamaegigas, the content of ABA conjugates, predominantly the glucose ester of ABA (ABA-GE), is low without distinct fluctuations of ABA-GE. Roots contained less ABA-GE than submerged leaves (Schiller et al. 1997). Conjugated ABA compounds have been regarded earlier as an indicator of stress history of plant tissues. They are deposited in the vacuoles where no further degradation is possible. This is not the case in Chamaegigas, which can release excess ABA to the surrounding medium. Deposition into the vacuoles is not necessary.

6.4.4 Distribution of ABA Between the Plants and the Pool Water

Apart from cycles of serious desiccation Chamaegigas is also exposed to drastic pH fluctuations, reaching alkaline pH values up to 12. We must, therefore, expect serious losses of ABA to the pool water considering that ABA distributes within Chamaegigas according to the anion trap concept (Hartung and Slovik 1991; Slovik et al. 1995). A significant loss of ABA during the afternoon hours might be a serious problem, especially because during this time pools usually dry up. Chamaegigas, however, seems to be well adapted to these conditions. Rosettes preloaded with labelled ABA lost significantly less ABA to the medium than a mesophytic terrestrial rosette (V. locusta) when transferred to an alkaline medium (pH 10).

With an increase of the external pool water pH by one pH unit, ABA in the pool water increased less than twofold (Schiller et al. 1997). According to the Henderson–Hasselbalch equation a tenfold increase should be expected. Uptake of external ABA was strongly pH dependent with high uptake rates under acid conditions, indicating that external ABA can be taken up during the night. Membranes of Chamaegigas roots must have a remarkably low permeability coefficient for undissociated ABA. Additionally one cannot exclude the existence of an effective hypodermal apoplastic barrier, which also could slow down ABA loss to the medium drastically (Freundl et al. 2000). This, together with a high cytosolic pH of nearly 7.6 (Schiller et al. 1998a), could explain the low external ABA concentration, which is below those predicted by computer simulations (Slovik et al. 1995) and found in soil solutions under different crops (Hartung et al. 1996).

In conclusion, the role of ABA in Chamaegigas is not in regulating stomatal aperture as in most other plants. Rather, the most sensitive ABA biosynthesis and the maintenance of high tissue ABA concentrations point to its essential function, e.g. in triggering metabolic processes (see below).

6.5 Dehydrins

Fully hydrated submerged leaves of Chamaegigas synthesise proteins that show similarities with the ABA-inducible desiccation-related proteins of C. plantagineum (Bartels et al. 1990), the dehydrins. Even after 4 weeks of optimal hydration, the dehydrin amount is high. In fully hydrated roots, however, dehydrins are not detectable. When hydrated roots were treated with ABA dehydrins were formed, as it was the case when roots desiccated. (The function of dehydrins is described in Chaps. 14 and 16.)

6.6 Carbohydrates

Poikilohydric plants exhibit high amounts of carbohydrates, predominantly sucrose, fructose and glucose, but also glucopyranosyl-β-glycerol, trehalose and arbutin (M. flabellifolia, Bianchi et al. 1991) and octulose in C. plantagineum where it is converted during desiccation to sucrose (Bianchi et al. 1991, 1992; Norwood et al. 1999, 2000; Scott 2000). In Chamaegigas, besides sucrose (140–180 μmol g−1 dw) stachyose (approx. 180–250 μmol g−1 dw) also becomes very dominant. Raffinose is present, however, at a relatively low level (approx. 20 μmol g−1 dw). Glucose and fructose are substantially higher in leaves (180–200 and 60–120 μmol g−1 dw, respectively) than in roots (20–30 μmol g−1 dw). During dehydration, the monosaccharides of the leaves decrease together with increasing stachyose and sucrose content (Fig. 12.5). Heilmeier and Hartung (2001) suggested that glucose and fructose are incorporated into sucrose and stachyose. In roots, stachyose content is similar to leaves, and the changes of the amounts of sugars mentioned above during desiccation are far less distinct.

Fig. 12.5
figure 5_12

Levels of fructose, glucose, sucrose and stachyose in submerged leaves of Chamaegigas inrepidus as a function of decreasing leaf water content (modified after Heilmeier and Hartung 2001)

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In air dry tissues, the total content of soluble carbohydrates of roots and submerged leaves ranged from 14 to 26% with stachyose (roots) and sucrose (leaves) as the dominant sugars. In roots, stachyose can account for 14% of dry weight. ABA treatment caused soluble carbohydrates to rise in roots, especially as far as sucrose is involved (Schiller 1998).

The function of sugars such as raffinose or stachyose or other galactosyl-sucrose oligosaccharides is the suppression of crystallisation of protoplastic constituents and the promotion of glass formation at low water content (Bruni and Leopold 1991; seed embryos). At a glassy state, a liquid has a high viscosity. Chemical reactions are slowed down, and interactions between cell components are prevented. Therefore, a glassy state is highly stable and ideal to survive desiccation. Furthermore, these raffinose family oligosaccharides (RFOs) may serve as storage carbohydrates for immediate regrowth of floating leaves and flowers after rewatering (C. plantagineum; Norwood et al. 2000).

7 Breeding System and Genetic Diversity in Chamaegigas Populations

As an endemic plant Chamaegigas grows in an extremely small area of central Namibia. Compared to plants of a wide distribution, such populations suffer from genetic impoverishment, i.e., a small number of polymorphic gene loci and a loss of alleles. Inbreeding within small populations and genetic drift may cause a reduced degree of heterozygosity and a fixation of harmful alleles. Additionally the isolated occurrence of Chamaegigas on inselbergs of approximately 25 km distance may reduce genetic exchange via seed dispersal and pollen transfer drastically. Thus, restricted genetic diversity should severely limit the potential for adaptation in Chamaegigas to its stressful environmental conditions.

However, molecular genetic investigation by means of AFLP-marker (amplified fragment length polymorphism) has shown a surprisingly high genetic diversity within the plants of one inselberg, even within one pool (Durka et al. 2004). This high genetic diversity is very likely due to pollination by four insects, two bee species ( Liotrigona bottegoi and Apis mellifera) and mainly two beetle species of the genus Condylops (C. erongoensis and a newly identified species) (Woitke et al. 2006). The zygomorphous and slightly protandric flowers show a typical insect pollination syndrome: they are distinctly coloured, intensively scenting and provide a rich floral reward (abundant pollen grains), but they do not produce any nectar. Rather, dense layers of trichomes (400–1,600 per mm2) can be found on the lower lip, similar to well-known oil flowers. However, there are no indications that Chamaegigas lives in an oil flower/oil-collecting bee symbiosis (Woitke et al. 2006). Pollination experiments indicated that Chamaegigas is a predominantly outcrossing species, and a large number of pollen could be found both on the wild bee and beetle species (Durka et al. 2004).

In conclusion, this endemic species does not show any genetic impoverishment. The high genetic variability may be a result of high UV radiation at high altitude, which should result in many mutations and a high gene flow both within inselbergs by pollinating insects and among inselbergs due to seed dispersal by wind or animals (Heilmeier et al. 2005). This may provide the genetic basis for a successful adaptation of Chamaegigas to its extreme habitat conditions.

8 Concluding Remarks

Chamaegigas is often considered to represent the most spectacular resurrection plant (Gaff and Giess 1986; Hartung et al. 1998) as it (1) shows the most rapid recovery after rewetting among all poikilohydric angiosperms, and (2) possesses two types of mature leaves with contrasting drought tolerance on the same plant. Therefore, this species has been considered to be well adapted to its natural habitat, shallow, only temporarily water-filled rock pools. However, as shown here, desiccation is only one aspect of a multi-dimensional complex of environmental stress conditions, which also include low partial pressures of carbon dioxide during the day when plants are flooded, drastic diurnal oscillations in pH and low nutrient contents of the pool water, high temperatures and solar radiation, especially in the ultraviolet range. Thus, both acquisition and conservation of resources such as carbon and nitrogen when they are scarce and protection of cellular and molecular structures from damage due to dehydration, alkaline, heat and UV stress are essential for the survival of this tiny plant at its extreme habitat.

Similar complex environmental stress conditions also influence other resurrection plants such as C. plantagineum and X. humilis, which grow in the direct vicinity of Chamaegigas bearing outcrops. Except pH fluctuations and CO2 availability, very similar environmental stresses (temperature, solar radiation, unfavourable pH of the soil solution and extreme nutrient deficiency) act on those plants as well. Salt stress may be even an additional problem. Growth conditions of poikilohydric study plants, grown in glasshouses or growth cabinets, may necessarily have to be adapted accordingly. More extremely tissues cultures, as they are often used in the case of C. plantagineum, grow under conditions that are extremely far from any reality, even if they may have been treated with osmotica.

In a recent opinion article, Marris (2008) has shown convincingly that molecular biology, the use of transgenic plants, and gene technology have not brought significant gain and improvement for increase of yield and performance of crops that are cultivated in arid regions. One reason may be that in many of these studies, the complexity of drought tolerance has not been considered sufficiently. To engineer successful transgenic crops not only drought-responsive genes should be considered, but also those of ABA metabolism, of transporters of nitrogenous compounds, of enzymes involved in cell wall biochemistry and membrane integrity, of enzymes involved in the acquisition of DON, of enzymes involved in salt and alkaline pH tolerance and pH homeostasis and of enzymes and metabolites involved in detoxifying reactive oxygen species. Research with plants such as Chamaegigas that have developed a complex system of strategies to survive the extremely harsh conditions can teach us the necessary direction of future research.