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Water availability is one of the factors severely limiting the productivity of agricultural crops in arid and semi-arid areas. Irrigation is the current strategy to overcome water shortage. However, the scale of problems of soil salinization associated with irrigation schemes is considerable and continuing to grow (Läuchli & Lüttge, 2002). Genetic engineering of drought-tolerant crop plants therefore provides a promising alternative approach for the management of areas where usable fresh water is limited. Substantial progress in this field can be expected if the genes and the physiological processes contributing to drought tolerance are identified in plants growing naturally on dry stands. These plants have evolved various, very efficient mechanisms to cope with drought. Thus, their investigation at the molecular and membrane level through whole-plant responses up to the ecosystem level may pave the way for the development of drought-tolerant crops.
Among the great diversity of drought-tolerant plants occurring in arid regions, the poikilohydric resurrection plant Myrothamnus flabellifolia is one of the most interesting candidates (Canny, 2000). This plant is a woody, multi-stemmed shrub that grows up to 1–2 m high in shallow soils on rocky outcrops in southern Africa. Upon drought, the leaves close like tiny inverted sunshades and become small brown rolls that furl very tightly to the branches. After rain, the leaves unfold and display their green adaxial surfaces within about 1 d, even after extremely long periods of drought. While the molecular biology, biochemistry and physiology of drought tolerance of M. flabellifolia have been studied in detail (Gündel, 1968; Hoffmann, 1968; Vieweg & Ziegler, 1969; Bewley & Krochko, 1982; Suau et al., 1991; Goldsworthy, 1992; Wilson & Drennan, 1992; Bianchi et al., 1993; Drennan et al., 1993; Sherwin & Farrant, 1996; Hartung et al., 1998; Farrant et al., 1999; Scott, 2000), the mechanistic aspects of the refilling process of the dry xylem elements have been addressed by very few groups (Child, 1960; Gaff, 1977; Goldsworthy, 1992; Sherwin et al., 1998).
Recently, 1H nuclear magnetic resonance (NMR) imaging, as well as light and electron microscopy studies on branches of M. flabellifolia have shown that an assembly of driving forces including root pressure and capillary forces as well as xylem surface tension and osmotic pressure gradients is involved in axial water ascent and radial refilling of the tissue cells of the resurrection plant (Schneider et al., 1999, 2000b; Wagner et al., 2000; Zimmermann et al., 2001). For the interactive operation of the various forces during water lifting, conditioning of the xylem elements by lipids seems to be of extremely high relevance. Controlled phase separation of lipids and water and lipid disposal within the xylem elements during dehydration are apparently of equal importance for the following rehydration/dehydration cycle.
Refinement of our current knowledge of xylem refilling and consolidation of the inferences drawn previously call for a spatial analysis of the lipid composition, distribution and mobility throughout the entire branch and the corresponding local interplay of lipids with water upon wetting. At present, data are available only for the internodes (Schneider et al., 1999, 2000b; Wagner et al., 2000; Zimmermann et al., 2001). By contrast, our knowledge about the nodal regions of the branch, including leaf traces, short shoots and leaves is poor. Understanding of the strategies adopted by M. flabellifolia to allocate water most efficiently also deserves – among other things – a detailed examination of the breakdown and disposal of lipids in conducting and nonconducting xylem elements.
As demonstrated here, substantial information about the putative role of xylem lipids in withstanding drying and facilitating subsequent rehydration could be obtained by light and electron microscopy and in particular by the numerous complementary variants offered by NMR spectroscopy and imaging (Kuchenbrod et al., 1995, 1996, 1998; Volke & Pampel, 1995; Bentrup, 1996; Manz & Callaghan, 1997; Manz et al., 1997, 1999; Meininger et al., 1997; Volke et al., 1997; Pampel et al., 1998; Rokitta et al., 1999; Seymour et al., 1999; Wagner et al., 2000; Wistuba et al., 2000; Peuke et al., 2001; Zimmermann et al., 2001). In contrast to the microscopy techniques, the noninvasive NMR methods allowed measurements on lipid composition, mobility and distribution on the same branch both in the presence and absence of water.
The experimental evidence given here corroborates the hypothesis that axial water rise and water spreading to the leaves and other tissue parts is under the control of lipids arising from their uneven distribution, mobility and composition. The results also support our previous view (Schneider et al., 2000b) that a combination of several methods is required for investigations of such a specialised plant system in order to avoid misinterpretations.
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- Materials and Methods
The results obtained by TEM, as well as by various NMR spectroscopy and imaging techniques differing greatly in the level of spatial resolution and acquisition of lipid parameters, have provided an integrated view about the important role of lipids in xylem and cell conditioning for dehydration and rehydration of the branches of M. flabellifolia. This species has obviously developed very economic strategies based on lipid distribution, composition and mobility, in order to use water efficiently and to minimize transpirational water loss.
The xylem of the internodes and nodes of the branch occupies at least 80% of the cross-sectional area. About half of the xylem can be assigned to the conducting pathway (Wagner et al., 2000). This region is separated from the nonconducting region by a concentric ring-shaped area of variable thickness (c. 50–750 µm) and low 1H signal intensity as identified in dry branches by NMR imaging. This ‘black ring’ arose from an ‘NMR anomaly’ induced by pronounced differences in the lipid content and density of the rays between this area and the adjacent conducting/nonconducting regions.
Both the conducting and nonconducting xylem regions contained a similar distribution of lipids within the ray cells, which yielded a uniformly low 1H NMR background signal. In the nonconducting area the intervessel pits were mostly completely clogged by lipid inclusions. The majority of the lumena of the xylem elements were free of lipids. However, extremely high lipid concentrations were found at the pith periphery and, interestingly, in some tracheid assemblies. Three-dimensional 1H NMR imaging reconstructions showed that these tracheid assemblies were not randomly arranged. They formed a fragmented hollow cylinder made up of ‘lipid pieces’– at least throughout the 2-cm long branch regions used for imaging. The physiological role of this peculiar structure is not clear at present. It seems likely that it represents a relict of a continuous cylindrical assembly of lipid-rich tracheids that served as a protection against complete water loss during extreme drought, particularly in the juvenile plant since ‘fragmented lipid rings’ are found only in the central xylem area. The finding of occasionally up to three of these cylindrical structures can also be taken as support for this assumption, because extremely dry periods are not unusual in the southern part of Africa. Disruption and associated fragmentation of these ‘protecting sheaths’ apparently occurred during internodal elongation. This explains straightforwardly the finding that the average size of the fragments was much larger in the nodes than in the internodes, and that the lipid fragments occasionally exhibited reflected images of edge curvature. Lipid filling of these tracheid assemblies is apparently achieved by the outermost cell layers of the branch located in the region of the cambium and/or cortex. This is suggested by NMR images of some dry branches collected from sites of extremely high ambient temperature and irradiation as well as of low relative humidity (Schneider et al., 1999). These occasionally showed unusually high lipid signal intensities in the outermost tissue region.
Although this certainly deserves further investigation, it seems clear that the lipid-rich tracheid/vessel assemblies in the leaf traces are obviously used for the restriction of water supply to the leaves. The NMR investigations suggested that only a few – if any – xylem elements were water-conducting. The majority of the xylem elements were unable to conduct water, a result which contradicts the current view about the function of leaf traces (Esau, 1977). Nonwettability was obviously achieved by thick composite lipid wall linings (which extended into the pits) and by additional filling of the pits with bulk lipids. The reduced conducting area explains the recent observation (Schneider et al., 2000b) that upon water supply to the cut end of the air-dry branches, leaf recurving always occurs with a delay of a few minutes after arrival of the water front at a certain height. Together with the morphological peculiarity of leaf furrow-located stomata (Schneider et al., 1999), reduction of the conducting area in the leaf traces seems to be the strategy of M. flabellifolia to minimize water loss by evaporation. The refilling studies by means of 1H NMR spectroscopy support this view. The main refilling phase was finished after about 40 min, in agreement with previous NMR imaging data (Wagner et al., 2000). However, owing to the higher sensitivity of NMR spectroscopy a subsequent, very slow refilling phase could be resolved, which obviously reflected compensation of water evaporation from tissue cells and/or cell wall material.
The resurrection plant M. flabellifolia apparently uses a similar strategy to Bulnesia sarmientoi to counteract excessive transpirational water loss under extreme environmental conditions. This tree, which grows up to 14-m tall, rooting in highly saline water at fairly high ambient temperatures (up to 40°C) facilitates axial water ascent at the expense of water supply to the leaves by clogging of a large part of the xylem of twigs at intermediate heights with mucopolysaccharides and proteins (Zimmermann et al., 2002). These NaCl-binding compounds are apparently optimal for the generation of hydraulic resistances when the plant is faced with salinity, but not with drought.
For M. flabellifolia the central problem is the reactivation of the xylem in the conducting area for water ascent after drought. As shown here, the xylem lipid linings as well as the bulk lipid inclusions of the pits and tracheid edges disintegrate upon rehydration. Lipid blebs and granular remnants are formed, which are presumably the precursors of the innumerable lipid bodies found in the lumena of water-filled xylem elements (Schneider et al., 1999; Wagner et al., 2000). An additional source for the formation of the lipid bodies upon wetting may be lipid secretion into the xylem by the ray cells, which are found in extraordinarily high abundance in M. flabellifolia (Carlquist, 1976). Because of the numerous pits between the ray cells and the xylem elements (see earlier) the ray cells can be regarded as contact cells. Such cells are known to release metabolites (such as lipids, sugars, proteins, etc.) into the adjacent xylem elements (Sauter et al., 1973; Esau, 1977). The finding that the ray cells of the ‘black ring’ area contain less lipid than those in the adjacent nonconducting and conducting areas could be taken as an indication for the release of lipids into the neighbouring xylem elements. This assumption would straightforwardly explain the transformation of conducting xylem elements in the ‘black ring’ area into nonconducting ones during seasonal growth.
The crucial point is the penetration of the lipid lining by water upon wetting required for axial water ascent and radial spreading, respectively. Mandatory for this process is apparently the thickness as well as the composition of the lipid lining of the xylem elements and pits. In the nonconducting area, the inner walls of the xylem elements and pits are covered by a very thick (sometimes more than 300 nm) lipid lining consisting of alternating thin (about 20 nm) and thick (about 60–80 nm) layers of high and low TEM contrast, respectively. Wetting and subsequent breakdown of these composite lipid linings upon contact with aqueous fixation solutions was never observed. This suggests that the thick, composite lipid linings contain a large amount of neutral, nonwettable lipids, which are presumably concentrated in the 60–80-nm-thick layers. This conclusion can be drawn from the observation that in the conducting area the inner walls of the xylem elements and pits are exclusively covered by a single 20-nm-thick layer which is water-wettable. The high TEM contrast of this layer, even in the absence of osmium tetroxide staining, indicates the presence of phospholipids, as mentioned earlier. The 13C MAS spectroscopy on air-dry branch wood also provided strong evidence for significant amounts of phospholipids. The value of the self-diffusion coefficient of 1.6 × 10−12 m2 s−1 deduced from the 1H NMR PGSTE experiments on dry branches is also typical for these compounds (Lindblom & Wennerström, 1977; Lindblom et al., 1977; Volke et al., 1994a; Galle & Volke, 1995). A major component of the phospholipids seen in the 13C MAS spectra seemed to be phosphatidyl choline (Fig. 1). The 13C MAS spectrum of Fig. 1 shows many other signals which must be attributed to lipids or phospholipids (e.g. phosphatidylethanolamines). However, they could not be identified unambiguously because of signal overlapping. The high resolution of the MAS spectra was achieved at the expense of spatial resolution. The possibility can therefore not completely be excluded that the signals recorded in the 13C MAS spectra and in the 1H NMR PGSTE experiments resulted from the rays or, more likely, from both compartments.
Phospholipids are known to bind significant amounts of water (Crowe & Crowe, 1984; Cornell et al., 1974; Gawrisch et al., 1978, 1992, 1995; Arnold et al., 1981; Volke et al., 1982). The self-diffusion coefficients deduced from 1H NMR PGSTE experiments performed on hydrated branches in axial and radial direction are consistent with this finding. As mentioned earlier, self-diffusion coefficients of 4.5 × 10−11 m2 s−1 and 2.7 × 10−10 m2 s−1 are typical for water bound to lipids or interacting with the polar regions of phospholipids (Volke et al., 1994a; Lindblom & Wennerström, 1977; Lindblom et al., 1977). Furthermore, the rapid gold sol penetration of the lipids of the conducting area can only be explained if phospholipids or, more generally speaking, amphiphilic lipids are involved (taking into account that the pit lipids exhibited only a low TEM contrast compared with the xylem linings).
The interactions of phospholipids (or related amphiphilic lipids) with water at the molecular level and the breakdown of phospholipid layers are well understood (Arnold et al., 1979; Crowe & Crowe, 1984; McIntosh, 1996; Huang & Epand, 1997; Gutberlet et al., 1998). Some of the phospholipids, especially phosphatidyl choline and phosphatidylserine, form crystalline lamellar structures in the dry state. Upon water binding to the polar heads the lamellar phase turns over into the nonlamellar inverted hexagonal II (HII) phase if the dynamic molecular shape of the lipids assumes a noncylindrical geometry. Whereas phosphatidylethanolamines readily form HII phases upon contact with water, other types of phospholipids (e.g. phosphatidyl cholines), even when fully hydrated, require additional factors like changes in temperature, fatty acid composition or electrostatic interactions to force them into the HII phase (Huang & Epand, 1997; Gutberlet et al., 1998). The tubes or micelles formed by the HII structure are water-conducting, thus facilitating penetration of water into the lipid layer. The HII structure is unstable: with increasing water penetration multilamellar liposomes or – in the presence of some fatty acids – lipid bodies are formed (partly by fusion). The phase transitions of the phospholipids are accompanied by characteristic changes in 31P NMR spectra (Cullis et al., 1983; Crowe & Crowe, 1984; Smith & Ekiel, 1984) which could also recently be resolved by preliminary experiments on branches of M. flabellifolia (F. Volke & B. Manz, unpubl. data).
The kinetics (as well as the temperature) of the phase transitions of pure phospholipids depend on the lipid composition and charge distribution within the lipid molecules, but may also change when other compounds, such as trehalose and sucrose, are present (Crowe & Crowe, 1984; Crowe et al., 1984a; Smith & Ekiel, 1984; Hoekstra et al., 1997; Wolfe & Bryant, 1999; Koster et al., 2000; Oliver et al., 2002). These carbohydrates are well-known to protect membranes against dehydration and were also found in high amounts in cells of M. flabellifolia and other desiccation-tolerant organisms (Crowe & Crowe, 1984, 2000; Crowe et al., 1984a,b; Gadd et al., 1987; Bianchi et al., 1993; Drennan et al., 1993; Oliver et al., 2002). Preliminary studies on M. flabellifolia have shown that the xylem sap of hydrated plants also contains trehalose, sucrose and other lipid-interacting sugars in variable amounts. Taking the relatively slow dehydration process into consideration one can assume that spatial accumulation and depletion, respectively, results in an uneven distribution of these sugars throughout the xylem elements of the conducting area. The associated differences in lipid conditioning could easily explain why axial water ascent in dry branches is favoured initially by only a few elements (Schneider et al., 2000a,b; Wagner et al., 2000; Zimmermann et al., 2001). The involvement of carbohydrates in xylem lipid conditioning certainly deserves further investigation. The focus of the future research must also include elucidation of the mechanisms protecting M. flabellifolia plants in the field against repetitive drought. Under laboratory conditions the branches of the resurrection plant lose their ability to refill entirely after the first cycles of rehydration and dehydration (Wagner et al., 2000). Therefore, analysis of sugars and other solutes which are secreted into the xylem sap during hydration and during the first phase of dehydration is of equally high importance. This knowledge and the findings reported here and in previous publications will facilitate the identification of the involved genes – an important step on the long way towards genetically engineered, drought-tolerant crops. Knowledge of protecting carbohydrates and their interaction with phospholipids will also certainly lead to novel strategies in cryopreservation of mammalian and plant cells. Generation of artificial anhydrobiosis in mammalian and plant cells is of great biomedical and biotechnological interest because the compromising effects of freezing and thawing on cells can be minimised or completely eliminated (Beattie et al., 1997; Wolfe & Bryant, 1999; Crowe & Crowe, 2000). Technologies for the injection of balanced mixtures of lipids, disaccharides and other related compounds into cells without adverse side-effects are available (Zimmermann & Neil, 1996). Thus, generation of artificial anhydrobiosis seems to be a goal that can soon be realized.