Possible Role of Pectin-containing Mucilage and ... - Semantic Scholar

Annals of Botany 101: 277–283, 2008 doi:10.1093/aob/mcm089, available online at www.aob.oxfordjournals.org

Possible Role of Pectin-containing Mucilage and Dew in Repairing Embryo DNA of Seeds Adapted to Desert Conditions Z HE N Y IN G HU A NG 1 , IVA N B O U B R I A K 2, 3 , DA P HN E J . O S B O R N E 4 , M IN G DO N G 1 AND Y I T Z C H A K G U T T E R M A N 5, * 1 Laboratory of Quantitative Vegetation Ecology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, P.R. China, 2Institute of Cell Biology and Genetic Engineering UAS, Kiev, 03143, Ukraine, 3Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, UK, 4Oxford Research Unit, The Open University, Foxcombe Hall, Boars Hill, Oxford OX1 5HR, UK and 5Jacob Blaustein Institute for Desert Research and Department of Life Sciences, Ben-Gurion University of the Negev, Sede Boker Campus, 84990, Israel Received: 31 January 2007 Returned for revision 22 February 2007 Accepted: 9 March 2007 Published electronically: 11 May 2007

† Background and Aims Repair of damage to DNA of seed embryos sustained during long periods of quiescence under dry desert conditions is important for subsequent germination. The possibility that repair of embryo DNA can be facilitated by small amounts of water derived from dew temporarily captured at night by pectinaceous surface pellicles was tested. These pellicles are secreted during early seed development and form mucilage when hydrated. † Methods Seeds of Artemisia sphaerocephala and Artemisia ordosica were collected from a sandy desert. Their embryos were damaged by gamma radiation to induce a standard level of DNA damage. The treated seeds were then exposed to nocturnal dew deposition on the surface of soil in the Negev desert highlands. The pellicles were removed from some seeds and left intact on others to test the ability of mucilage to support repair of the damaged DNA when night-time humidity and temperature favoured dew formation. Repair was assessed from fragmentation patterns of extracted DNA on agarose gels. † Key Results For A. sphaerocephala, which has thick seed pellicles, DNA repair occurred in seeds with intact pellicles after 50 min of cumulative night dew formation, but not in seeds from which the pellicles had been removed. For A. ordosica, which has thin seed pellicles, DNA repair took at least 510 min of cumulative night dewing to achieve partial recovery of DNA integrity. The mucilage has the ability to rehydrate after daytime dehydration. † Conclusions The ability of seeds to develop a mucilaginous layer when wetted by night-time dew, and to repair their DNA under these conditions, appear to be mechanisms that help maintain seed viability under harsh desert conditions. Key words: Adaptation strategy, Artemisia, desert dew, DNA repair, pectin, pellicle, seed survival.

IN TROD UCT IO N The ability of seeds of most temperate species to cease DNA replication in the embryo and then undergo a subsequent dehydration to less than 5 % moisture content with no permanent cellular damage or genomic impairment permits long-term seed survival for decades or even centuries (Osborne, 1980, 2000). Water is a pre-eminent requirement for metabolic re-activation and germination. If the water potential is too negative to initiate full germination, seeds can still initiate certain biochemical processes that allow embryo cells to progress to the S1 stage of the first cell cycle. Such seeds will undergo important processes of pre-germination, including those crucial for maintaining their viability, for example DNA repair (Elder et al., 1987; Boubriak et al., 2001). These processes can improve subsequent performance under suboptimal conditions for germination. For this reason, their induction is widely practised by industry. It is known as ‘priming’ or ‘preconditioning’ because the embryos remain tolerant to desiccation and can be re-dried without harm in the short-term (Heydecker et al., 1973; Heydecker, 1977; Heydecker and Coolbear, 1977). * For correspondence. E-mail [email protected]

A related phenomenon may apply to seeds of many annuals and shrubs of desert regions that have evolved special survival mechanisms. A limited number of species have adapted to shifting dunes or to the more stable sands, such as those to be found in the Mu-Us Desert in China or in the Negev Desert of Israel. These species have attracted attention because of their potential for undergoing opportunistic DNA repair as part of their long-term survival strategy that necessarily involves survival for many years on or near the surface of dry sand and exposed to intensive UV irradiation. In such dehydrated conditions, the embryos of dry seeds slowly degenerate, enzyme proteins lose function and, in particular, the DNA of nuclear and mitochondrial genomes suffer progressive cleavage and other in situ damage from non-enzymatic methylation, oxidation and dimerization of nucleotides (Osborne and Boubriak, 1994; Osborne, 2000). They can also suffer from random fragmentation of chromosomal sequences (Cheah and Osborne, 1978). Unless the activity of the enzymes of the DNA repair complex (a and b polymerases, DNA ligase) (Elder et al., 1987; Osborne et al., 2002) remain sufficiently functional before germination is fully initiated, the embryo cells will fail to recover a functional genome and the seed dies. The maintenance of an

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Huang et al. — Pectin Pellicles, Dew and Seed Embryo DNA Repair

operational DNA repair complex is therefore an essential requirement for long-term survival in the dry state. Experiments were conducted linking repair of DNA to the presence of a water-absorbing polysaccharide pellicle deposited on the outside of the seeds (achenes) of Artemisia sphaerocephala, an important pioneer plant occurring in moving sand dunes (Wei and Tu, 1980) and of Artemisia ordosica, a species restricted to stable sands (Huang and Gutterman, 1999a, 2000). The pellicles form a mucilaginous layer after wetting (e.g. by dew forming at night when temperatures fall markedly in the desert). The findings indicate that this night-time trapping of water is sufficient to support repair to damaged DNA even though hydration levels are too low for germination. In this way, the integrity of the genome can be retained over the long term (Elder and Osborne, 1993). It is known that as little as 30 min is sufficient for repair of accumulated DNA lesions to take place (Boubriak et al., 1997). Here the extent to which hydrated mucilage can maintain genome integrity during dew formation was examined under actual night-time desert conditions and before the seed is again dehydrated in the hot desert daytime air. M AT E R I A L S A N D M E T H O D S Location of seed collection

Artemisia sphaerocephala and A. ordosica flower from July to August. During development, achenes are protected by the inflorescences and thus protected from wetting by rain until after dispersal. The achenes begin to mature from mid September to early November and are subsequently dispersed by wind and adhere to sand grains by the mucilaginous layer that forms on the achene surface when wetted. Mature achenes of A. sphaerocephala and A. ordosica were collected from dry unopened inflorescences in December, 2002, from natural populations in the Mu-Us sandy desert in Yulin (388060 N, 1078300 E; 1288 m a.s.l.), China. Here the annual rainfall varies from 100 to 450 mm and falls mainly from July to September with most night-time dew deposition occurring in August and September (Zhang, 1986, 1994). In the laboratory, inflorescences were manually shaken to detach the achenes, which were stored in a closed cotton bag at 4 8C.

Following irradiation, all embryos examined showed substantial damage to DNA as assessed by alkaline DNA electrophoresis (Elder and Osborne, 1993), which then became the basis for setting-up DNA repair assessments in vivo. Dew experiment in the desert

These experiments were made at the Jacob Blaustein Institute for Desert Research, Ben-Gurion University, in the Negev Desert highlands of Israel (308510 N, 348460 E; 460 m a.s.l.) where annual rainfall varies from 29 to 165 mm and occurs mainly from October to April (Gutterman, 2002). Night-time dew deposition is most frequent from July to October (Zangvil, 1996). Dew deposition conditions in the Negev are similar to those found in the shifting sand deserts in China (Zhang, 1986; Kidron, 2005). After radiation treatment, two replicated samples (50 per set) of cleaned and intact lots of A. sphaerocephala seeds and of intact A. ordosica seeds were weighed and spaced out individually on the surface of Negev desert sand previously rinsed with distilled water. The weighed sand was held in Petri dishes to a depth of 3 mm. The Petri dishes were arranged on trays and the edges of trays were protected by sticky glue against predators during nightly exposures to high humidity and dew deposition. These experiments were carried out in the field between 2000 h and 1000 h from 24 July to 2 August, 2003. The dishes were set up at the same time each evening for nine nights. From 0500 h on the next morning (after the time of maximum dew deposition), trays were repeatedly returned to the laboratory for repeated re-weighings every 15 min to determine rates of water loss while on the desert floor. Weighings were continued until the seeds had returned to their original air-dry weight (Fig. 1). After final weighing, the seeds were returned to the desert. Empty dishes were used to take account of any changes in sand or dish weights, although none proved large enough to affect the results. Not all nights were dew nights, so some sets of seeds were left out on consecutive nights to accumulate a period of time of .90 % relative humidity (RH) in the external air. These were the conditions when dew formed

Pellicle removal and gamma irradiation

A quantified dose of gamma radiation was used to induce the same amount of single-strand DNA breaks into embryo DNA (Elder and Osborne, 1993; Boubriak et al., 1997). Prior to this, pellicles were removed from half the seeds of A. sphaerocephala as follows. The seeds are small (1.0 – 1.5 mm long) and were held in water for exactly 0.5 h and then rubbed rapidly on filter paper to remove the pellicle as confirmed under the light microscope. Batches of cleaned and intact A. sphaerocephala seeds and intact seeds of A. ordosica were then gamma-irradiated (1000 Gy) using a 137Cs, RX 30/55 M Irradiator (Gravetom Industries, Gosport, Hampshire, UK) to induce a standard amount of damage to the DNA of embryo cells.

F I G . 1. Time-course data of morning water loss by 50-mg samples of seeds of Artemisia sphaerocephala (with or without pellicles) and Artemisia ordosica from the time of maximum night hydration by dew until the samples regained their original weight with the early morning rise in temperature. Seeds were kept on the desert floor during drying. An example is shown of one of 11 dew-loss measurements.


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in the desert. These accumulated periods of dew ranged from 50 to 390 min. Throughout these procedures, the monitoring station of the Desert Meteorology Unit of Jacob Blaustein Institute recorded relative humidity (Fig. 2) and temperature. Temperature and relative humidity measurements were taken every 10 min via a thermohygrograph (W. Lambrecht GmbH Gottingen, Germany). Data recording software was prepared by ‘Nimbus’, Israel. After the night-time exposures and once air dry, all the weighed individual sets of seeds were enclosed in Eppendorf tubes for DNA fragmentation analysis. DNA extraction and DNA fragmentation analyses

After the last desert exposure, the dry seeds were analysed for DNA fragmentation patterns. In this way the extent of any repair of the original irradiation-induced DNA damage was determined. Two replicates of each weighed sample of A. sphaerocephala seeds (70 mg with pellicle; 50 mg minus pellicle) and 60 mg of A. ordosica seeds were each ground in a mortar with three successive additions of dry ice. The resulting powder was extracted by using the Wizard Genomic DNA Kit (Promega Corp., Madison, WI, USA) as recommended by the manufacturer, except that nuclei lysis solution was used throughout for extraction. The DNA extracts were purified by re-suspension and ethanol precipitation and quantified by micro-cell UV analysis at 260 nm and by Pico GreenTM (Molecular Probes Europe, Breda, the Netherlands) fluorescence. Equal DNA loadings, 2 mg for A. sphaerocephala and 4 mg for A. ordosica, were applied to 0.8 % agarose gels and subjected to electrophoresis at 55 mA for 45 min. DNA was visualized with ethidium bromide and the gels scanned and recorded on a UVP data image analyser (Spring Scientific Ltd, New York, USA) (Figs 3 and 4).

F I G . 3. Mobility scans of DNA extracted from seeds of Artemisia sphaerocephala following gamma-irradiation treatments of 1000 Gy when dry and then an exposure to monitored periods of night-time with dew deposition in the desert of the Negev. Lane 1, DNA marker, 125– 23130 bp; Lane 2, dry seeds, pellicle removed; Lane 3, dry seeds, pellicle removed, irradiated; Lane 4, dry seeds, pellicle intact; Lane 5, dry seeds, pellicle intact, irradiated; Lane 6, seeds with pellicle removed after five cold desert nights (no dew); Lane 7, seeds with pellicle intact and after five cold desert nights (no dew); Lane 8, seeds with pellicle removed and after 50 min of night-time dew deposition; Lane 9, seeds with pellicle intact and after 50 min of night-time dew deposition; Lane 10, seeds with pellicle removed and after 120 min of night-time dew deposition; Lane 11, seeds with pellicle intact and after 120 min of night-time dew deposition; Lane 12, seeds with pellicle removed and after 390 min of night-time dew deposition; Lane 13, seeds with pellicle intact and after 390 min of night-time dew deposition; Lane 14, DNA marker, 500–5000 bp.

Scanning electron microscopy

Dry achenes or achenes that had been exposed to 340 min of one night dew and dried to their original weight were examined for structural changes following hydration of

F I G . 2. Daily rhythm of relative humidity of the air of the Negev desert during exposure of seeds of Artemisia sphaerocephala and Artemisia ordosica on the desert floor. Total time of dew deposition (i.e. duration above 90 % of relative humidity in minutes) is shown on the three nights when dew was deposited on the seeds.

F I G . 4. Mobility scans of DNA extracted from seeds of Artemisia ordosica following gamma-irradiation treatments of 1000 Gy when dry and then an exposure to monitored periods of night-time with dew deposition in the Negev desert. All pellicles were intact. Lane 1, DNA marker, 1000– 12 000 bp; Lane 2, intact dry seeds; Lane 3, irradiated dry seeds; Lane 4, seeds exposed to 50 min of night-time dew deposition; Lane 5, seeds exposed to 120 min of night-time dew deposition; Lane 6, seeds exposed to 390 min of night-time dew deposition; Lane 7, seeds exposed to 510 min of night-time dew deposition; Lane 8, DNA marker, 100–1500 bp.

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F I G . 5. Scanning electron micrographs of sections of seeds of Artemisia sphaerocephala (A, C) and Artemisia ordosica (B, D). A and B show dry seeds at the time of shedding. C and D show seeds after exposure to 340 min of dew deposition and subsequent re-drying. The pellicle layer of A. sphaerocephala is shown to collapse when dried after wetting with dew (C). This does not affect its ability to reform mucilage on further re-wetting. P, pellicle; T, testa; Cot, cotyledonary cells.

the pellicle and later re-drying. Achenes of A. sphaerocephala and A. ordosica were first halved transversely with a sharp scalpel to expose a cross-section of the pellicles (Fig. 5). For scanning electron microscopy, material was mounted on grids and coated with gold in a gold sputter at reduced pressure. Specimens were then examined and photographed in a Zeiss SUPRA 55VP (Carl Zeiss Ltd, Jena, Germany) analytical scanning electron microscope. R E S U LT S Repeated weighings of duplicate sets of seeds, set out on Negev sand, in weighed Petri dishes allowed estimations of maximum night-time dew deposition on the seeds as well as the rates of drying through the early morning until the seeds regained their original weight. The experiments were performed over 10 d, which included some nights when no dew was formed although temperatures decreased. Only dew deposition resulted in pellicles swelling to form a mucilaginous covering. Without pellicle, seeds of A. sphaerocephala returned to the original weight within 1.5 h of dewing, whereas those with intact pellicles took at least 3 – 4 h (Fig. 1) indicating a larger water-carrying capacity of seeds with pellicles in place. Seeds of A. ordosica, which have only thin pellicles, took only 2 h to dry back to their normal daytime weight. Figure 2 shows relative humidity during the Negev experiments. Dew deposition took place during three nights out of nine when there was no wind and

clear skies and continued as long as relative humidity was 90 %. The sum of the exposures to dew-forming conditions were 0, 50 (one night’s dew), 120 (one night’s dew), 390 (two nights’ dew: 50 min þ 340 min) and 510 (three nights’ dew: 50 min þ 120 min þ 340 min) min. Seeds with dew condensation could easily be recognized by a glistening of the hydrated mucilage. Mobility scans of extracted DNA (Fig. 3) show that the gamma-irradiation of dry seeds effectively fragmented embryo DNA as indicated by a long smear (Lane 3). However, only 50 min of dew was needed to permit some repair of this damage (short smear in Lane 9) in seeds of A. sphaerocephala with intact pellicles. Repair was more complete when dew deposition was extended to 120 (Lane 11) or 390 min (Lane 13). If the pellicle was removed, little or no repair occurred (Lanes 8, 10 and 12) with the long smears indicating extensive DNA fragmentation that could not be rescued by dew deposition. In comparison with those for A. sphaerocephala, scans of DNA from intact A. ordosica seeds (Fig. 4) revealed that DNA repair of gamma-irradiation-induced fragmentation takes much longer. At least 510 min of cumulative night-time with dew was needed to achieve even partial recovery of DNA integrity (Lane 7). This is in line with the relatively thin pellicular covering of this species and its corresponding lower water-retaining capacity. Scanning electron micrographs (Fig. 5) illustrate how the pellicle covering is deposited during seed formation, the pellicle layer of A. ordosica being about one-third the thickness of that of A. sphaerocephala (see also Huang and

Huang et al. — Pectin Pellicles, Dew and Seed Embryo DNA Repair Gutterman, 2000). In addition, the pellicle structure of A. sphaerocephala is shown to collapse after exposure to dew and subsequent dehydration. However, these collapsed pellicles were able to reform the mucilage layer when re-wetted. DISCUSSION Some of the most common annual and perennial plant species occurring in the Saharo-Arabian and Irano-Turanian phytogeographical regions of the Negev desert of Israel (Feinbrun-Dothan, 1978, 1986; Zohary, 1966, 1972) have seeds which develop a mucilaginous layer on their outer surface shortly after wetting (Gutterman, 1993, 2002). The mucilage is generated from pectin-rich pellicles on the testa or pericarp layers of oneseeded fruits or seeds (Zohary, 1962). The various species have different strategies and mechanisms of seed dispersal and germination. Some have their seeds dispersed by wind at the beginning of the summer and others by rain (Gutterman et al., 1967; Baskin and Baskin, 1998; Gutterman, 2002). In some of the latter species, seeds adhere to the soil surface and germinate during rain dispersal, as in Blepharis spp. (Gutterman et al., 1967; Witztum et al., 1969). Seeds of certain other species stick to the soil surface and may germinate in rains the following year or even after many years. In some cases, seeds stick to the soil surface by means of a mucilaginous layer, such as in Artemisia sieberi (Gutterman, 1993), Plantago coronopus, Carrichtera annua, Anastatica hierochuntica (Gutterman and Shem-Tov, 1996, 1997) and Artemisia monosperma (Huang and Gutterman, 1998). Seeds with a mucilaginous layer derived from a thick pellicular coating can attain their maximum water-absorbing capacity very quickly, for example within 2 h in Artemisia monosperma (Huang and Gutterman, 1999b; Huang et al., 2000). The seed, especially the upper parts, remains hydrated for longer (Huang and Gutterman, 1999b) as indicated in Fig. 1. DNA fragmentation studies show that this allows time for repair of damaged DNA in the seed embryo before the seeds re-dry in the morning sun (Figs 3 and 4). Dew deposition for a few minutes is all that is needed for extensive repair to take place. In the central part of the Negev desert, there are approximately 190 nights with each year when dew forms for 2 – 12 h (Evenari et al., 1982). A repair mechanism by re-wetting from any source has been termed ‘preconditioning’ or ‘priming’ by Heydecker and colleagues (Heydecker et al., 1973; Heydecker, 1977; Heydecker and Coolbear, 1977). This is known to improve the speed and percentage of germination when appropriate conditions are present for actual germination, as shown in Artemisia monosperma (Huang and Gutterman, 1999b). Such a repair mechanism is likely to increase the longevity of seeds that are on the sand surface and exposed to desert nights with dew, as shown in Artemisia sieberi by Evenari and Gutterman (1976). There is considerable published evidence linking DNA repair or the lack of it with the level of germination success. For example, a feature associated with loss of

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viability in dry seed is the increasing loss of integrity of nuclear DNA with increasing levels of low-molecular-weight oligonucleotide fragments (Cheah and Osborne, 1978; Elder et al., 1987; Elder and Osborne, 1993; Leprince et al., 1996). When Secale cereale (rye) embryos that are non-viable are imbibed for 2.5 h, the extent of fragmentation of DNA shows a significant increase, pointing toward the continuous activity of endonuclease cleavage in the absence of DNA repair (Cheah and Osborne, 1978; Elder et al., 1987). Embryos of imbibed dormant Avena fatua (oat) also show an efficient repair of radiation-induced lesions in the DNA (Boubriak et al., 1997), and this repaired DNA remains stable when embryos are re-dried. For both Secale cereale and Avena fatua seeds, the failure to restore genetic integrity to the genome on rehydration of an embryo whose DNA is damaged by desiccation is likely to be a major factor in determining desiccation tolerance (Boubriak et al., 1997). The ability to repair DNA when embryos are rehydrated is therefore critical to maintaining a functional genome. In addition, the stability of the DNA on desiccation and the capacity for repair of DNA on rehydration are considered to be integral components of a total desiccation tolerance mechanism (Elder et al., 1987; Osborne et al., 2002). In the present study, the DNA of Artemisia seeds was examined following DNA damage via gamma-irradiation. These seeds were exposed to normal nights in the Negev desert highlands with dew deposited naturally on the soil surface. The remarkable stability of the pellicle to enzymatic hydrolysis (Wei and Tu, 1980) and the lack of evidence that it provides a food source at germination, suggested that it might play a reliable long-term role in controlling hydration of the embryo and also in fixing Artemisia seeds to sand particles when the pellicles are moistened to form mucilage (Gutterman, 2002; Huang et al., 2004). Genes involved in the synthesis of mucilage by developing seeds of Arabidopsis have been reported (Western et al., 2000; Penfield et al., 2001; Haughn and Chaudhury, 2005). It should be emphasized, however, that the cells involved in pectin polysaccharide synthesis in Arabidopsis are not the same as those in Artemisia. In Arabidopsis, epidermal cells of the outer cell layers of the seed integument produce mucilage between the primary wall and the plasma membrane (Western et al., 2004). In Artemisia, the pectin is secreted outside the pericarp of an achene, and so forms an outer pellicle coat to the whole seed (Fig. 5). DNA repair occurring during imbibition of water in other species is known to remain stable after embryos are again dehydrated (Elder and Osborne, 1993; Boubriak et al., 1997). The present experiment shows that without their pellicle covering and resulting mucilage production on wetting, seeds of A. sphaerocephala were unable to repair DNA that had been damaged by prior gamma-irradiation. This effect arises because the mucilaginous layer keeps the embryo sufficiently hydrated for long enough to allow DNA repair processes to proceed even though germination per se is not induced. Seeds of A. ordosica, which colonizes stable but not shifting dune sand, have only a thin pellicle

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and hold dew for several hours less than those of A. sphaerocephala. Accordingly, there was far less effective DNA repair of prior gamma-irradiation damage in A. ordosica. These findings help to understand the ecological importance of mucilaginous layers when wetted for seed survival in the desert. Understanding the unusual pellicles of Artemisia species and their role in DNA repair may offer new opportunities for the priming and coating of high-value seeds to protect them from damage during fast desiccation after aerial seedling treatment and during later hydration and desiccation in field conditions, especially once the desiccation intolerance stage has set in. Coating seeds with appropriate hygroscopic material is therefore suggested for aerial seedlings during ecological restoration of degraded ecosystems, such as shifting desert sand dunes. ACK N OW L E D G E M E N T S We thank The Royal Society of London and The Chinese Academy of Sciences for supporting this joint research project. Z.H. acknowledges the financial support by an innovative group research grant (30521002) from the National Natural Science Foundation of China, the Key Basic Research and Development Plan of China (2007CB106802), Key Project of CAS (KZCX2-XB2-01) and National Natural Science Foundation of P.R. China (30000021, 30570281). We thank Professors Carol and Jerry Baskin for critical review of this manuscript, and Dr Heather Davies of the Electron Microscopy Unit, The Open University, UK, for her valuable advice. L I T E R AT U R E CI T E D Baskin CC, Baskin JM. 1998. Seeds – ecology, biogeography, and evolution of dormancy and germination. San Diego: Academic Press. Boubriak I, Kargiolaki H, Lyne L, Osborne DJ. 1997. The requirement for DNA repair in desiccation tolerance of germinating embryos. Seed Science Research 7: 97–105. Boubriak I, Kostuyk O, Naumenko V, Matorina N, Grodzinsky D. 2001. Irradiation as a test-factor revealing dark DNA repair capability of seeds. Cytology and Genetics 35: 54–59. Cheah KSE, Osborne DJ. 1978. DNA lesions occur with loss of viability in embryos of ageing rye seed. Nature 272: 593–599. Elder RH, Osborne DJ. 1993. Function of DNA synthesis and DNA repair in the survival of embryos during early germination and in dormancy. Seed Science Research 3: 43– 53. Elder RH, Dell’Aquila A, Mezzina M, Sarasin A, Osborne DJ. 1987. DNA ligase in repair and replication in the embryos of rye, Secale cereale. Mutant Research 181: 61–71. Evenari M, Gutterman Y. 1976. Observations on the secondary succession of three plant communities in the Negev desert, Israel. I. Artemisietum herbae albae. In: Hommage au Prof. Chouard P, Jacques R, eds. Etudes de Biologie Vegetale. Paris: C.N.R.S. Gif sur Yvette, 57–86. Evenari M, Shanan L, Tadmor N. 1982. The Negev, the challenge of a desert, 2nd edn. Cambridge, MA: Harvard University Press. Feinbrun-Dothan N. 1978. Flora Palaestina. Part Three—Text. Jerusalem: Israeli Academy of Sciences and Humanities. Feinbrun-Dothan N. 1986. Flora Palaestina. Part Four—Text. Jerusalem: Israeli Academy of Sciences and Humanities. Gutterman Y. 1993. Seed germination in desert plants. New York: Springer. Gutterman Y. 2002. Survival strategies of annual desert plants. New York: Springer.

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