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Journal of Arid Environments 70 (2007) 183–194
Journal of Arid Environments www.elsevier.com/locate/jaridenv
Anatomical, morphological and metabolic acclimation in the resurrection plant Reaumuria soongorica during dehydration and rehydration Y.B. Liua, G. Wangb, J. Liuc, X. Zhaoa, H.J. Tana, X.R. Lia, a
Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, PR China b Key Laboratory of Arid and Grassland Agroecology of Ministry of Education, School of Life Sciences, Lanzhou University, Lanzhou 730000, China c Department of Plant Pathology, Physiology and Weed Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA24060, USA Received 30 December 2005; received in revised form 1 November 2006; accepted 15 December 2006 Available online 26 February 2007
Abstract Reaumuria soongorica (Pall.) Maxim., a short woody shrub found widely in semi-arid areas of China, can survive severe desiccation of its vegetative organs. We studied the anatomical, morphological and metabolic acclimation of R. soongorica in leaf and stem tissues during desiccation and in stems upon rewatering. During dehydration, the mesophyll and choloroplast ultrastructure were disturbed in leaves, but not in stems. Water storage tissues were rich in osmotic substances in both organs. Upon rewatering, osmophilic globules in stems disappeared and a repair process was observed in phloem. Highly specialized stomata, which are ring-shaped and raised, were found to expand in hydrated stems and dried leaves. The many glands present on leaves were assumed to play a role in desiccation tolerance. Nuclear magnetic resonance (NMR) data showed that sucrose concentrations increased with stress, which probably resulted in higher concentrations of osmotic substances in water storage tissues. Malate and proline, which accumulated in stems during water loss, may play a major role in osmoregulation. In conclusion, the stem was able not only to maintain the structural integrity of mesophyll cells and chloroplasts during dehydration, but also to repair the phloem structure on rewatering. The stem also accumulated metabolic products that
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play roles in osmoregulation. All these findings indicate that the stem is an essential organ for water deficit survival. r 2007 Elsevier Ltd. All rights reserved. Keywords: Anatomy; Choloroplast ultrastructure; Desiccation; Metabolic products; Nuclear magnetic resonance
1. Introduction The ability of vascular flowering plants to tolerate desiccation is scarce. Resurrection plants are a group of plants that can tolerate prolonged protoplast desiccation conditions and revive upon rehydration (Gaff, 1971, 1977). To survive in dry habitats, resurrection plants have to overcome a number of stresses caused by dehydration, such as oxidative stress, destabilization or loss of membrane integrity, and mechanical stress (Vicre´ et al., 2004b). Different resurrection plants may utilize several different physiological and biochemical mechanisms to adapt to desiccation and regain normal metabolic status upon rehydration (Bewley and Krochko, 1982; Navari-Izzo and Rascio, 1999). A number of morphological modifications associated with dehydration have been observed as adaptations in resurrection plants to minimize damage caused by light and free radical stress in dry tissues. Water stress produces major changes in the morphology and anatomy of desiccation-tolerant plants (Vander Willigen et al., 2003). Dehydration causes curling or folding of the leaves and considerable reductions in cell volume in the resurrection plant Craterostigma wilmsii (Farrant, 2000; Sherwin and Farrant, 1998). In another resurrection plant, Xerophyta viscosa, dismantling of chloroplasts was observed, which may be necessary to prevent excess light absorption. On the contrary, retention of chloroplast integrity was found in C. wilmsii and enabled a rapid recovery of photosynthesis upon rehydration (Sherwin and Farrant, 1998). Metabolites, as the terminal products of cellular metabolism, are valuable indicators of how a biological system responds to environmental changes (Fiehn, 2002). Among these, carbohydrates act as a protection against dry-induced damage to cell membranes, which is a common mechanism utilized by all desiccation-tolerant organisms. Changes in carbohydrate levels during drought are essential in desiccation tolerance because carbohydrates play important roles in many physiological processes, such as photosynthesis, respiration, and nutrient uptake and transport (Bruni and Leopold, 1991; Vertucci and Farrant, 1995). Sucrose, raffinose and trehalose are also considered to be involved in glass formation and to interact protectively with membrane phospholipids. We report here on a set of experiments performed with Reaumuria soongorica (Pall.) Maxim., a short woody shrub with vegetative organs that can survive desiccation. During the dry season, the plant desiccates and the leaves abscise. Even several weeks later, the stem is still able to reactivate and develop new leaves upon rainfall. Therefore, it is qualified as a resurrection plant. Our major aim was to follow the anatomical and morphological changes in leaf and stem tissues during dehydration of R. soongorica and in stems upon rewatering, and to investigate the contribution of these changes to desiccation tolerance. Some low-molecular-weight metabolic substances that are believed to be involved in the stabilization and composition of cell structures were also analyzed to explore their potential roles in the resurrection phenomenon.
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2. Material and methods 2.1. Plant material Two-year-old R. soongorica plants were used. Twenty plants were transplanted into 20-cm-diameter pots and transferred to the Botanical Garden of Lanzhou University and acclimated for 4 months, keeping them fully watered until the beginning of the experiment in August 2005. One set of plants was dehydrated by withholding water at air temperature and ambient photoperiod, whereas another set was watered daily under the same conditions during the entire experimental period. Leaves and stems were harvested from fully hydrated leaves (C), and on days 7 (D1), 10 (D2), and 15 (D3) after the beginning of dehydration. Rehydration was started immediately after the plant leaves died. Stems were collected at 24 and 72 h (R1 and R2 stages, respectively) after rewatering of the plants. Leaf water potential was determined on freshly cut leaves using a WP4 Dewpoint Water Potential Meter (Decagon Devices, Inc, Pullman, WA, USA). For microscopy analysis, samples were selected from young leaves and stems of comparable size, and then fixed immediately in a phosphate buffer containing 3% glutaraldehyde for 24 h. All the remaining samples were stored in liquid nitrogen. Experiments were performed in triplicate for each sampling date. 2.2. Light and electron microscopy Samples were washed twice for 10 min in phosphate buffer (0.1 M) at pH 7.2, and then postfixed with 1.0% OsO4 in the same buffer overnight. After post-fixing, samples were washed with the same phosphate buffer and dehydrated in increasing concentrations of ethanol. Pre-inclusion of samples in freshly prepared resin at 35 1C (24 h) was followed by final inclusion in acetone/ epon812 (1:1) (12 h at 45 1C and 24 h at 65 1C). For light microscopy, semi-thin sections (1.0 mm) were stained with 1% toluidine blue and observed. For transmission electron microscopy (TEM), ultra-thin sections (70 nm) were obtained using an ultramicrotome (LKB-V, Sweden) and stained with 3% uranyl acetate followed by lead citrate before observation with a TEM (JEM-1230, Japan). For scanning electron microscopy (SEM), the samples were fixed, post-fixed and dehydrated as above, dried using JFD-310 apparatus (Japan), covered with 20 nm of gold, and observed in a JSM-6380 (Japan) microscope. 2.3. Analysis of metabolite products using NMR methods 2.3.1. NMR sample preparation Samples were prepared according to Manetti et al. (2004) with a modification. Each sample was weighed (0.8 g fresh weight) and then ground into a fine powder in liquid N2 and maintained in a liquid N2 bath during the pulverization procedure. An aliquot of 3 ml of methanol/chloroform (2:1) was added to the powder. The powder was then stirred and 1 ml of chloroform and 1.2 ml of water were added. All samples was stored at 4 1C for 1 h and then centrifuged at 6000g for 20 min at 4 1C. The resulting upper hydro-alcoholic and lower chloroform phases were separated. The extraction procedure was performed twice on the pellet for quantitative extraction. After the second extraction, the hydroalcoholic phases obtained from both extractions were pooled, dried under N2 flux, and stored at 80 1C prior to analysis.
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2.3.2. NMR data collection For NMR spectra, dried samples were dissolved in 0.5 ml of 0.5 mM TSP solution in D2O PBS buffer (pH 7.4) to avoid chemical shift changes due to pH variations. Dissolved extracts were transferred to a 5-mm NMR tube. NMR spectra were recorded on Varian Mercury 300-MHz instrument (USA). Spectra were referenced to TSP [sodium salt of 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid] at a final concentration of 0.5 mM. TSP was used as a reference for both chemical shifts (0.00 ppm) and quantitation of signals (Defernez and Colquhoun, 2003). Processing of spectra was carried out using Mestre-C software. The spectra were scaled to fix the area of the TSP signal to a value of 10. For each tissue analyzed, triplicate spectra were recorded. Resonances due to sucrose, glucose, proline, malic acid and citrate were identified by their chemical shifts. 3. Results 3.1. Desiccation and recovery After 15 days of desiccation, the leaf water potential (WP) of R. soongorica had dropped to 15.9 MPa (Fig. 1). Then the leaves of the stressed plants and some of the fine terminal twigs died. New leaves developed from the old twigs within 10–15 days upon rewatering. 3.2. Anatomical studies The anatomical effects of dehydration on leaves and dehydration–rehydration on stems were analyzed by light microscopy of cross-sections (Fig. 2). In the leaf tissues of control plants (Fig. 2a and d), the palisade parenchyma of the mesophyll was formed by several layers of cells and had limited intercellular spaces. Abundant water-storage tissues filled up the space between neighboring vascular bundles. Progressive dehydration treatment caused severe damage to the mesophyll structure and organization (Fig. 2b and c), in comparison to mesophyll in the control plants. The mesophyll was highly disorganized with large and irregularly shaped air spaces. It is interesting that the large water-storage cells formed
0
WP(MPa)
-5 -10 -15 -20 -25 0
3
6
9
12
15
Time after last watering (days)
Fig. 1. Leaf water potential (WP) during desiccation of R. soongorica. Time ‘0’ represents the non-desiccated controls. Data represent mean7SE, n ¼ 3.
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Fig. 2. Light microscope photography showing transverse sections of leaves (a–f) and stems (g–k) in R. soongorica during dehydration. The images are shown in 10 (a–c, g–k), bars ¼ 0.1 mm, and 100 (d–f) magnification, bars ¼ 10 mm. (a, d, g), fully hydrated plant (WP, 0.8 MPa); (b, e, h), desiccated plant in D2 stage (WP, 7.2 MPa); (c, f, i), desiccated plant in D3 stage (WP, 15.9 MPa). (j) Rewatered plant in R1 stage (24 h after rewatering); (k) rewatered plant in R2 stage (72 h after rewatering). P, palisade mesophyll; VB, vascular bundle; WS, water storage tissue; OG, osmophilic globules.
many osmophilic globules (Fig. 2e). In plants of the D3 stage (Fig. 2c and f), although the water storage cells were filled with osmotic substances, they showed more severe damage to the leaf structure than for D1 and D2 plants.
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In stems, numerous water-storage tissues formed more osmophilic globules between the phloem and xylem during dehydration (Fig. 2g–i). There was no visual damage to the stem anatomy during dehydration. Even in plants of the D3 stage, the integrity of the whole structure of the stem was maintained and only the air spaces increased in the central cylinder of the xylem. Upon rehydration, in stem tissues of R1 stage (Fig. 2j), the osmophilic globules disappeared completely. The phloem was separated from the xylem and was full of irregular air spaces. In the R2 stage (Fig. 2k), however, the two parts recombined, suggesting that the stems may be involved in the repair mechanisms involved upon rehydration. 3.3. Morphological changes Fig. 3 shows surface views of the epidermis from hydrated leaves, dry leaves and dry stems. In leaves, SEM examination of drought-treated samples revealed that the epidermis of D3-stage leaves (Fig. 3b) was highly folded in comparison with that from hydrated plants (Fig. 3a). Glands were observed in the epidermis of both control and dehydrated leaves. Nevertheless, a few small, ring-shaped, raised and widely opened stomata were found in leaves in the D3 stage (Fig. 3b and d) but not in control leaves (Fig. 3a and c). In stems, there were many such stomata trapped in the epidermal folds of the control plants (Fig. 3e), but not in the dried (Fig. 3f) and rewatered plants (Fig. 3g). Unlike the control and rewatered plants, plants in the D3 stage showed large papillae on the epidermis of stem tissues (Fig. 3f). 3.4. Chloroplast ultrastructure Cell and chloroplast structures were studied by TEM. In the leaves of control plants, chloroplasts in mesophyll cells were attached to the cell wall and covered most of it (Fig. 4a). They exhibited a typical chloroplast structure and ellipsoid shape, with clearly evident thylakoid membranes organized in stromal but not in granal membranes (Fig. 4c). However, in dehydrated plant leaves (Fig. 4b), mesophyll cells suffered severe damage and most chloroplasts were disordered. Drought stress resulted in complete damage of the chloroplast structure and shape (Fig. 4d). The integrity of the outer membrane and the internal network of thylakoid membranes were almost completely lost. However, chloroplasts from dehydrated stem mesophyll (Fig. 4h) were structurally similar to those from control plants (Fig. 4g) and showed typical stromal thylakoids, like any photosynthetically active tissue. No damage was observed in the subcellular structure of the stems during dehydration (Fig. 4f). Vacuoles and mitochondria were visible in mesophyll cells in both hydrated and dehydrated plants (Fig. 4e and f). 3.5. Metabolite analysis Table 1 shows the levels of some metabolic products that changed significantly during dehydration and rehydration. Among the sugars, fructose appeared to be at very low concentrations after desiccation and rewatering (data not shown). The levels of sucrose and glucose varied dramatically among the different tissues and over time. Sucrose and glucose concentrations increased with stress and decreased upon rewatering in stems.
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Fig. 3. Scanning electron microscopy micrographs showing the surface views of leaves (a–d) and stems (e–g) in R. soongorica during dehydration. (a) Surface view of the control plant, 350, bar ¼ 50 mm; (b) surface view of the desiccated plant in D3 stage (WP, 15.9 MPa), 350, bar ¼ 50 mm; (c) glands of the control plant at higher magnification, 750, bar ¼ 20 mm; (d) largely opened stomata of the desiccated plant in D3 stage at higher magnification, 1400, bar ¼ 10 mm; (e) surface view of the control plant, 3000, bar ¼ 5 mm; (f) surface view of the desiccated plant in D3 stage (WP, 15.9 MPa), 1200, bar ¼ 10 mm; (g) surface view of the rewatered plant in R2 stage (72 h after rewatering), 1600, bar ¼ 10 mm; g, glands; st, stomata; p, papilla.
However, in leaf tissues, sucrose levels increased during the first stage of drought stress and remained at a high level, while the concentrations of glucose declined all through during the drought period. The response of proline to increased stress was similar to that of sucrose in the stems. During prolonged severe desiccation, concentrations of proline
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Fig. 4. Transmission electron microscopy of mesophyll cells and chloroplasts of leaves (a–d) and stems (e–h) in R. soongorica during dehydration. (a) Mesophyll cell of the control plant, 5000, bar ¼ 1 mm. (b) Mesophyll cell of the desiccated plant in D3 stage (WP, 15.9 MPa), 4000, bar ¼ 1 mm. (c) Mesophyll chloroplast of the control plant, 60,000, bar ¼ 50 nm. (d) Mesophyll chloroplast of the desiccated plant in D3 stage, 40,000, bar ¼ 100 nm. (e) Mesophyll cell of the control plant, 25,000, bar ¼ 0.2 mm. (f) Mesophyll cell of the desiccated plant in D3 stage (WP, 15.9 MPa), 30,000, bar ¼ 0.1 mm. (g) Chloroplast of the control plant, 60,000, bar ¼ 50 nm. (h) Chloroplast of the desiccated plant in D3 stage, 60,000, bar ¼ 50 nm. Cp, chloroplasts; N, nucleolus; V, vacuoles; M, mitochondria.
increased and remained elevated during dehydration. Regarding organic acids, malate concentrations increased with stress in stems, but declined in dried leaves and rewatered stems. Citrate was not detectable in the leaves of control plants, but was evident at low concentrations in dehydrated plants, suggesting a pattern of increase during dehydration. In contrast, in stems, citrate gradually decreased and almost disappeared during desiccation and then increased upon rewatering.
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Table 1 Relative storage capacity (mg/g fresh weight) for some low-molecular weight metabolic substances in the leaves and stems of R. soongorica during dehydration and rehydration C
D1
D2
D3
R1
14.7 (1.72)a 16.5 (2.47)c
15.4 (2.12)a 21.9 (3.24)b
14.9 (2.34)a 37.1 (4.27)a
15.7 (2.14)c
6.9 (0.26)b
R2
Sucrose Leaf Stem
9.1 (1.32)b 8.7 (1.33)d
Glucose Leaf Stem
4.6 (0.72)a 2.7 (0.48)c
3.2 (0.54)ab 4.1 (0.42)c
2.5 (0.36)b 5.7 (0.34)b
0.6 (0.11)c 8.8 (0.21)a
Proline Leaf Stem
0.9 (0.12)d 1.3 (0.15)c
2.37 (0.13)c 1.97 (0.21)bc
4.3 (0.53)b 3.5 (0.62)b
13.4 (1.42)a 8.43 (0.93)a
nd d
nd d
Malate Leaf Stem
2.13 (0.06)a nd e
1.78 (0.35)a 2.32 (0.28)d
1.2 (0.69)b 5.4 (0.66)c
0.38 (0.84)c 9.84 (0.92)a
6.97 (0.92)b
2.31 (0.36)d
Citrate Leaf Stem
nd b 0.46 (0.04)a
0.05 (0.02)a 0.21 (0.05)b
0.04 (0.01)a 0.06 (0.02)cd
0.07 (0.06)a nd d
0.16 (0.05)cb
0.28 (0.09)b
5.1 (0.63)d
4.3 (0.28)bc
Data are the mean7SD; n ¼ 3. Standard deviations are given in parentheses. Letters represent the mean separation by Scheffe’s multiple range tests, showing comparisons in each parameter (Po0.05). C, fully hydrated plants (WP, 0.8 MPa); D1, drought-stressed plants (WP, 4.1 MPa); D2, desiccated plants (WP, 7.2 MPa); D3, desiccated plants (WP, 15.9 MPa); R1, rewatered plants (24 h after rewatering); R2, rewatered plants (72 h after rewatering). nd, not detected.
4. Discussion A combination of anatomical, morphological and metabolic adaptations may be critical for the acclimation of R. soongorica to arid desert environments. Leaves and stems accumulated many large osmophilic globules in water storage tissues when subjected to stress, but desiccation caused severe damage to the leaves, in which mesophyll cells were distorted and rich in air spaces. However, no destruction was observed in the phloem of stems. The cells showed tighter structural arrangement than those in control plants, except for cavities in the xylem. Osmophilic globules occupied the greatest part of the phloem. On rewatering, the well-organized phloem structure was disturbed and showed a repair process. We believe that the disappearance of osmophilic globules was due to their low osmotic potential. Upon rehydration, the osmophilic globules might have absorbed so much water from the external space that the membranes surrounding them burst. Micromorphological analysis revealed a high density of glands on the surface of both control and dehydrated leaves. Similar glands have previously been reported (Vicre´ et al., 2004a) in the resurrection plant C. wilmsii, and were assumed to play a role in desiccation tolerance. An unexpected finding was that drastic opening of the small, ring-shaped and raised stomata only showed on the surface of severely desiccated leaves and control stems. The density of stomata on the desiccated leaves was very low, while most stomata in control stems were trapped in epidermal folds. To maintain their internal water content,
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many xerophytes are rich in serum and develop stomata in local leaf epidermis depressions or in crypts (Wang and Wang, 1989). R. soongorica shows highly specialized stomata compared to other xerophytes. We assume that these raised stomata may be more sensitive to air moisture conditions. The guard cells may cave in the leaf epidermis when the stomata are closed. A major process for water economy is a reduction in transpiration by closure of stomata. However, in desiccated leaves of R. soongorica, stomata were largely open. Such a phenomenon was described for C. wilmsii (Vicre´ et al., 2004a). This indicates that R. soongorica maintains cell turgor by lowering the cell osmotic potential, closing stem stomata and opening a significant number of leaf stomata, probably maintaining CO2 uptake. Many large papillae were observed on the epidermis of desiccated stems, but their function is unknown and there is no report on this phenomenon in other similar species. Desiccation showed strong effects on the chloroplast ultrastructure in leaf tissues, including disruption of the outer membrane and stromal structure. No chloroplast structural damage was observed in stems. It is possible that the ability to maintain chloroplast integrity in stems allows rapid recovery of photosynthesis upon rehydration. This phenomenon has also been reported in the resurrection fern Polypodium polypoides (Muslin and Homann, 1992), the resurrection moss Tortula ruralis (Seel et al., 1992) and many homoiochlorophyllous resurrection plants such as Boea hygrometrica (Deng et al., 2000) and Ramonda serbica (Gesneriaceae) (Markovska et al., 1994). The chloroplasts of R. soongorica in both leaves and stems showed only stroma thylakoid but no grana, which might be correlated with the evolutionary history of R. soongorica in desert environments. To distinguish the accumulation of metabolites associated with osmoregulation from the simple concentration process accompanying water loss, NMR spectroscopy was selected in the present study. NMR spectroscopy can be used for analysis of the composition or metabolic profiling of plant tissues, which allows identification and quantification of the most abundant mobile metabolites and determination of the concentration changes caused by environmental disturbances (Bligny and Douce, 2001; Ratcliffe and Shachar-Hill, 2001). Plants in desiccated conditions reach low cell osmotic potential by accumulating solutes, such as carbohydrates, organic acids, amino acids, phenols, etc., to maintain cell turgor and thus cause opening of stomata (Chartzoulakis et al., 1999; Kameli and Losel, 1995; Patakas and Noitsakis, 1999). Sucrose, in addition to its ability to stabilize enzymes and cellular structures in the absence of water, can also vitrify the cell contents and stabilize the internal cell structure (Cowre et al., 1998). Some resurrection plants are capable of accumulating large amounts of sucrose in tissues (Scott, 2000). In R. soongorica, accumulation of sucrose probably results in stabilization of the cell structures. During dehydration, glucose levels increased in the stems, but decreased in the leaves, suggesting that glucose might be a temporary storage carbohydrate. The concentrations of some organic acids also varied significantly in hydrated and dehydrated plants. Malate, which often seems to increase under stress conditions in plants that display osmotic adjustment (Gebre and Tschaplisnki, 2002), decreased in R. soongorica leaves during dehydration. This may possibly be related to the disturbed photosynthetic apparatus. As an alternative end product of glycolysis, malate is considered a good indicator of photosynthetic capacity (Patonnier et al., 1999). Proline potentially plays roles in gene expression and antioxidative defense systems (Hare et al., 1999; Hong et al., 2000). Elevated levels of proline may assist in osmotic adjustment during desiccation.
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In R. soongorica, the processes involved in desiccation tolerance and recovery upon rewatering were achieved through complex structural and functional adaptations. The adaptational variations in morphology and anatomy observed from leaves to stems indicate that the stem is an essential structure for water-deficit survival and serves as a desiccation-tolerant and resurrection organ. Meanwhile, metabolic changes in the stem confirm that it may serve as a temporary storage organ during dehydration. Acknowledgment This work was supported by Natural Science Foundation of China (No. 40471006). References Bewley, J.D., Krochko, J.E., 1982. Dessication tolerance. In: Lange, O.L., Nobel, P.S., Osmond, C.B., Ziegler, H. (Eds.), Encyclopedia of Plant Physiology. New series, vol. 12B. Springer, Berlin, pp. 325–378. Bligny, R., Douce, R., 2001. NMR and plant metabolism. Current Opinion of Plant Biology 4, 191–196. Bruni, F., Leopold, A.C., 1991. Glass transition in soybean seed. Plant Physiology 96, 660–663. Chartzoulakis, K., Patakas, A., Bosabalidis, A.M., 1999. Changes in water relations, photosynthesis and leaf anatomy induced by intermittent drought in two olive cultivars. Environmental and Experimental Botany 42, 113–120. Cowre, J.H., Carpenter, J.F., Crowe, L.M., 1998. The role of vitrification in anhydrobiosis. Annual review of physiology 60, 73–103. Defernez, M., Colquhoun, I.J., 2003. Factors affecting the robustness of metabolite fingerprinting using 1H NMR spectra. Phytochemistry 62, 1009–1017. Deng, X., Hu, Z., Wang, H., Wen, X., Kuang, T., 2000. Effects of dehydration and rehydration on photosynthesis of detached leaves of resurrective plant Boea hygrometrica. Acta Botanica Sinica 42, 321–323. Farrant, J.M., 2000. A comparison of patterns of desiccation tolerance among three angiosperm resurrection plant species. Plant Ecology 151, 29–39. Fiehn, O., 2002. Metabolomics—the like between genotypes and phenotypes. Plant Molecular Biology 48, 155–171. Gaff, D.F., 1971. Desiccation-tolerant flowering plants in Southern Africa. Science 174, 1033–1034. Gaff, D.F., 1977. Desiccation tolerant vascular plants of Southern Africa. Oecologia 31, 93–109. Gebre, G.M., Tschaplisnki, T.G., 2002. Solute accumulation of chestnut oak and dogwood leaves in response to throughfall manipulation of an upland oak forest. Tree Physiology 22, 251–260. Hare, P.D., Cress, W.A., Van Staden, J., 1999. Proline synthesis and degradation: a model system for elucidating stress-related signal transduction. Journal of Experimental Botany 50, 413–434. Hong, Z., Lakkineni, K., Zhang, Z., Verma, D.P.S., 2000. Removal of feedback inhibition of D1-pyrroline-5carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiology 122, 1129–1136. Kameli, A., Losel, D.M., 1995. Contribution of carbohydrates and other solutes to osmotic adjustment in wheat leaves under water stress. Journal of Plant Physiology 145, 363–366. Manetti, C., Bianchetti, C., Bizzarri, M., Casciani, L., Castro, C., Ascenzo, G.D., Delfini, M., Cocco, M.E.D., Lagana, A., Miccheli, A., Motto, M., Conti, F., 2004. NMR-based metabonomic study of transgenic maize. Phytochemistry 65, 3187–3198. Markovska, Y., Tsonev, T., Kimenov, G., Tutekova, A.A., 1994. Physiological changes in higher poikilohydric plants—Haberlea rhodopensis Friv. and Ramonda serbica Panc. during drought and rewatering at different light regimes. Journal of Plant Physiology 144, 100–108. Muslin, E.H., Homann, P.H., 1992. Light as a hazard for the desiccation-resistant ‘‘resurrection’’ fern Polypodium polypoides L. Plant, Cell and Environment 15, 81–89. Navari-Izzo, F., Rascio, N., 1999. Plant responses to water deficit conditions. In: Pessarakli, M. (Ed.), Handbook of Plant and Crop Stress. Marcel Dekker Inc., New York, pp. 231–270. Patakas, A., Noitsakis, B., 1999. Mechanisms involved in diurnal changes of osmotic potential in grapevines under drought conditions. Journal of Plant Physiology 154, 767–774.
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