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Mrs Alitza Iglesias, Mrs. Sandra María Llanes, Mrs. Yudelmis de la Pera and Mr ... Cejas I, Méndez R, Villalobos A, Palau F, Aragón C, Engelmann F, Carputo D, ... Companioni B, Mora N, Díaz L, Pérez A, Arzola M, Espinosa P, Hernández M, ...

CryoLetters 34 (4), 413-421 (2013) © CryoLetters, [email protected]

BIOCHEMICAL CHARACTERIZATION OF ECUADORIAN WILD Solanum lycopersicum MILL. PLANTS PRODUCED FROM NONCRYOPRESERVED AND CRYOPRESERVED SEEDS Byron Zevallos1, Inaudis Cejas2, René Carlos Rodríguez3, Lourdes Yabor3, Carlos Aragón3, Justo González3, Florent Engelmann4, Marcos Edel Martínez3 and José Carlos Lorenzo3 1

Escuela Superior Politécnica Agropecuaria de Manabí Manuel Félix López (ESPAM), Campus Politécnico El Limón, Carrera de Ingeniería Agrícola, Calceta, Manabí, Ecuador, 2 Faculty of Agronomy, Universidad de Ciego de Ávila, Ciego de Ávila 69450, Cuba. 3 Laboratory for Plant Breeding, Centro de Bioplantas, Universidad de Ciego de Ávila, Ciego de Ávila 69450, Cuba. URL: 4 IRD, UMR DIADE, 911 Avenue Agropolis, BP 64501, 34394 Montpellier Cedex, 5, France. *Corresponding author e-mail: [email protected] Abstract This paper presents some of the effects of cryopreservation of wild Solanum lycopersicum Mill. seeds on the early stages of germination post liquid nitrogen exposure. Percentage of germination, conversion into plantlets and plant fresh mass were evaluated after cryostorage. Levels of chlorophyll pigments (a, b, total), malondialdehyde, other aldehydes, phenolics (cell wall-linked, free, and total) and proteins were determined. Peroxidase and superoxide dismutase activities were recorded. Liquid nitrogen exposure increased the percentage of seed germination at 5 days but at 7 days, the conversion into plantlets and the plant fresh mass were not statistically different between non-cryopreserved and cryopreserved samples. Several significant effects of cryopreservation were recorded at the biochemical level at 7 days of germination under controlled conditions. Highly significant effects due to liquid nitrogen exposure were observed in leaves: increased levels of peroxidase enzymatic and specific activities and cell wall-linked phenolics. Very remarkable effects were also recorded in roots: decreased contents of chlorophylls and cell wall-linked phenolics. Keywords: tomato; cryostorage; biochemical changes; phenotypic variation. INTRODUCTION With the unprecedented loss of valuable plant germplasm occurring globally, it becomes increasingly important to conserve seeds ex situ in genebanks (51). Seed storage is certainly the most effective and efficient method for ex situ conservation of plant genetic resources (38). Recommended optimal seed storage conditions include conservation in hermetically sealed containers, seed moisture content between 3–7% (fresh weight basis) depending on the species and a storage temperature of –18°C or below. Such conditions guarantee the retention of high levels of viability over extended time spans, possibly centuries (49). 413

The possibility that much longer periods of seed storage are attainable at ultra-low temperature using cryopreservation has been highlighted (49). Under cryopreservation, at -196°C, the plant material can be stored without alteration or modification for very long time periods. Moreover, cultures are stored in a small volume, protected from contamination, and require very limited maintenance (20). An earlier prediction carried out by Pritchard (46) suggested that seed longevity at cryogenic temperature could be about 175 times longer than at conventional seed bank temperature. More recently, cryogenic half-lives up to c. 3,400 years were predicted for lettuce seeds, based on experiments running for longer than 10 years (60). Comparable estimates of lettuce seed longevity at –18°C, based on the seed viability equation developed by Walters et al. (60), were approx 46–70 years, i.e. 74 times less than at cryogenic temperature. Therefore, as an extra insurance policy for conservation, the use of cryopreservation should be considered for all orthodox seeds, and one sub-sample of any accession systematically stored in liquid nitrogen (LN), in addition to the samples stored under classical genebank conditions (21, 48). The physiological state of samples before they are cryobanked has a strong impact on their long-term stability and viability (6). Moreover, physical and biochemical parameters of the plant material have an effect on its storability over long periods. Thermal-stress induced fractures of biological materials may cause serious damage to stored samples. Fractures typically occur in large organs such as whole seeds and are less common in cell suspensions and meristems. Most reports of physical cracking are found with animal organs (liver slices, veins, and arteries) rather than plant specimens (50). In the literature, there are many publications, which describe cryopreservation techniques (7, 19, 22, 54). By contrast, only a few studies have been carried out on the understanding of the biochemical effects of LN exposure and of the physiological changes occurring after LN storage of seeds (12, 21, 28, 29, 36, 57). In the genebank context and from an agronomic point of view, the effect of liquid nitrogen (LN) exposure on seed viability, germination, biochemistry, physiology and capacity for production of true-to-type plants should be tested for each plant material before using cryopreservation for long-term storage. Phenotypic assessment is probably the easiest way to detect any change following cryopreservation. In a recent work performed by our group on cryopreservation of Phaseolus vulgaris seeds, no phenotypic change was observed in seedlings recovered from cryopreserved seeds (11, 12). However, several significant effects were recorded at the biochemical level, including a decrease in protein and phenolics content and an increase in aldehyde contents in shoots, and a decrease in phenolics contents in roots. In general, roots were more affected by cryostorage compared to other organs. In this paper, we referred several times the work of Cejas et al. (12) because, to the best of our knowledge, it is the only research protocol similar to the one described here for tomato seeds. In the case of Solanum lycopersicum Mill., seeds are known to show orthodox storage behaviour (60) and to be tolerant to desiccation and LN exposure (55, 56). At present, the Polytechnic Agriculture and Cattle Husbandry University of Manabi (ESPAM, Ecuador) is conducting a research project aimed at collecting, characterizing and conserving the wild tomato (S. lycopersicum Mill.) genetic diversity occurring in Bolivar Canton (North-central section of the Manabi Province, 537.8 km2), genes of which could be useful for future breeding programs. Moreover, some accessions are rare or endangered. The project involves characterization of in situ environmental conditions where plants grow and ex situ phenotypic description and molecular marker analysis of collected samples. Furthermore, the establishment of a wild tomato seed cryobank is also planned. This paper presents some of the effects of cryopreservation of wild S. lycopersicum seeds on the early germination stages post LN storage. We studied several compounds related to a


wide range of important biochemical and physiological pathways associated with storage compounds, membrane and cell wall damage, and ROS generation depending of physiological processes such as germination, plant response to stress and photosynthesis (25, 31, 32, 43, 44, 45, 61). Percentage of germination, conversion into plantlets, plant fresh mass and protein concentration were evaluated. Levels of chlorophyll pigments (a, b, total), malondialdehyde, other aldehydes, phenolics (cell wall-linked, free, and total) were determined. Superoxide dismutase and peroxidase activities were also recorded. Moreover, such a study could provide a broad picture of the possible effects of cryopreservation on early stages of germination. Besides, these results show the evolution of these parameters in seeds during their storage for 14 days under normal storage conditions and under LN storage. To the best of our knowledge, such information on wild S. lycopersicum seeds has not been published to date.

MATERIALS AND METHODS After harvesting from the greenhouse, S. lycopersicum Mill. seeds (accession 56) were stored for 4 months at 4°C in the dark, in hermetically closed containers and in a controlled and constant environment. Seed moisture content (fresh weight basis) was determined by oven drying at 130°C for 1.5 h (34). Seeds with 12% moisture content (based on fresh weight (34) were used. One half of the seeds were placed in cryo-vials (volume: 2 ml; 50 seeds per cryo-vial) and immersed in LN for 2 weeks. The other half remained in the same conditions as described above (control treatment). Recovery of seeds from LN was performed according to Stanwood & Bass (56). From each treatment, seeds were randomly selected to perform the following steps of experimentation. Seeds (three replicates of 50 seeds per treatment) were placed in Petri dishes (Ø: 100 mm) with 15 ml distilled water for 7 days (dark, 27±1°C). Percentage germination (5 days), conversion into plantlets and plant fresh mass (7 days) were evaluated. Biochemical analyses of seedlings were performed at day seven of germination. Each biochemical determination was performed using three independent samples (100 mg each). Samples were finely ground in LN. Contents of malondialdehyde and other aldehydes (30); chlorophyll [a, b, total (45)]; phenolics [cell wall-linked, free, total (26)]; and proteins (8) were determined. Peroxidase (27) and superoxide dismutase (42) activities were also measured. Enzyme activities were also expressed on the basis of protein as specific activity. Seven days after treatment completion, samples for total protein, peroxidase and superoxide dismutase activity determination were immediately extracted in a buffer solution and stored at -20°C until quantification. Malondialdehyde, other aldehydes, chlorophylls and phenolic compounds were extracted and quantified immediately after fresh sample collection. Malondialdehyde and other aldehydes were quantified by a colorimetric method based on the reaction with thiobarbituric acid. Chlorophylls were extracted from plant material and directly quantified using a spectrophotometer. Phenolic compounds were extracted and then quantified using a spectrophotometer by a colorimetric method based on the reaction with Folin Ciocalteu reagent. Proteins were extracted in a buffer solution and quantified by a colorimetric reaction followed by spectrophotometer determination. Enzymes were extracted in buffer solutions and quantified by direct and continuous enzymatic methods on the spectrophotometer. The Statistical Package for Social Sciences (Version 17.0 for Windows, SPSS Inc.) was used to perform t-tests and compare results of the two treatments studied: non-cryopreserved and cryopreserved seeds (p≤0.05). Then the overall coefficients of variation (OCVs) were calculated as follows: (standard deviation/average) * 100. In this formula, to calculate the 415

standard deviation and average of the two treatments, we considered the average values of non-cryopreserved and cryopreserved seeds. Therefore, the higher the difference between the two treatments compared, the higher the OCV. OCVs were classified in three categories: for OCVs below 36%, the effect of cryopreservation of seeds was regarded as ¨low¨; from 36 to 72% as ¨medium¨, and above 72% as ¨high¨ (12). RESULTS LN exposure significantly increased the percentage of seed germination at 5 days, from 62.2 to 75.5% for non-cryopreserved and cryopreserved seeds, respectively (Table 1, Figure 1). However, at 7 days the conversion into plantlets and the plant fresh mass were not statistically different between these two treatments. Table 1. Percentage of germination, conversion into plantlets and fresh mass of Solanum lycopersicum Mill. plants from non-cryopreserved and cryopreserved seeds.

Noncryostored seeds 62.2 b 53.3 a

Cryostored OCV seeds (%)**

Percentage of germination (5 days) * 75.5 a 13.65 Percentage of conversion into plantlets 57.3 a 5.11 (7 days) * Plant fresh mass (mg) (7 days) * 12.4 a 12.8 a 2.24 * Results with the same letter are not statistically different (t-test, p>0.05). For statistical analysis only, percentages were transformed according to y´=2*arcsine(y/100)0.5. ** Overall coefficient of variation = (standard deviation/average)* 100. To calculate this coefficient, average values of non-cryopreserved and cryopreserved seeds were considered. The higher the difference between the two materials compared, the higher the overall coefficient of variation.

Several significant effects of cryopreservation were recorded at the biochemical level at 7 days of germination (Table 2). ¨High¨ OCVs were observed in leaves, with increased levels of peroxidase enzymatic and specific activities and cell wall-linked phenolics. ¨High¨ OCVs were also recorded in roots, with decreased chlorophyll (a, b, total) contents and cell wall-linked phenolics. The effects of cryostorage were classified as ¨medium¨ (according to their OCV) for the following indicators (Table 2): increased total content of phenolics in leaves; and higher levels of malondialdehyde and other aldehydes in roots. Seed LN exposure significantly decreased the total phenolics content in roots. LN exposure increased peroxidase enzymatic and specific activities and free phenolics content in shoots. Figure 1. Effect of cryopreservation of Solanum lycopersicum Mill. seeds 7 days after initiation of germination.


Table 2 Biochemical characterization of Solanum lycopersicum Mill. plants from non-

cryopreserved and cryopreserved seeds at 7 days after initiation of germination. Plant roots from nonfrom cryostored cryostored seeds seeds Total chlorophyll concentration (μg -1 * g FW) Chlorophyll a concentration (μg -1 * g FW) Chlorophyll b concentration (μg -1 * g FW) Malondialdehyde −1 content (μmol g * FW) Other aldehyde −1 content (μmol g * FW) Content of cell wall-linked −1 phenolics (mg g * FW) Content of free −1 phenolics (mg g FW) Total content of −1 phenolics (mg g * FW) Total protein −1 content (mg g * FW) Peroxidase −1 activity (U mg * FW) Peroxidase specific activity −1 (U mg of * protein) Superoxide dismutase activity −1 * (U mg FW) Superoxide dismutase specific activity −1 (U mg of * protein)

146.28 a

81.19 a

65.08 a

24.35 b

75.30 b

11.80 a

4.30 a

16.10 a

0.013 b

27.21 b

423.91 b

5.01 a

90.86 a

Plant shoots from nonfrom cryostored cryostored seeds seeds

38.22 b, OCV** 82.83% 21.78 b, OCV 81.60% 16.44 b OCV 84.38% 63.45 a, OCV 62.98% 216.07 a, OCV 68.33% 3.21 b, OCV 80.93%

290.26 a

3.90 a, OCV 6.90% 7.10 b, OCV 54.86% 0.015 a, OCV 11.20% 41.69 a, OCV 29.71% 557.05 a, OCV 19.19%

6.58 a

3.58 b, OCV 23.57% 55.04 b, OCV 34.73%

178.15 a

112.12 a

7.89 b

61.76 b

6.34 a

12.92 a

0.013 a

4.34 b

67.47 b

5.73 a

102.65 a

Plant leaves from nonfrom cryostored cryostored seeds seeds

241.51 b, OCV 12.96% 148.60 b, OCV 12.79% 92.90 b, OCV 13.26% 10.04 a OCV(%) 16.96 69.16 a, OCV 7.99% 2.97 b, OCV 51.19%

583.19 a

2.61 b, OCV 61.09% 5.58 b, OCV 56.11% 0.14 a, OCV 8.24% 11.58 a, OCV 64.28% 159.79 a, OCV 57.45%

3.35 b

6.11 a, OCV 4.56% 96.96 a, OCV 4.03%

218.17 a

365.01 a

45.74 a

507.55 a

3.22 b

6.58 b

0.025 a

2.50 b

19.70 b

8.60 a

78.01 b

581.12 a, OCV 0.25% 216.87 a, OCV 0.42% 364.25 a OCV 0.15% 46.33 a OCV(%) 0.91 471.91 b, OCV 5.15% 13.78 a, OCV 87.85% 4.57 a, OCV 21.78% 18.35 a, OCV 66.77% 0.023 b, OCV 7.53% 13.12 a, OCV 96.03% 115.12 a, OCV 100.08% 10.32 a, OCV 12.86% 104.21 a, OCV 20.34%

OCV ave = OCV ave = OCV ave = 49.32% 28.53% 32.31% *In each plant organ, results with the same letter are not statistically different (t-test, p>0.05). **Overall coefficient of variation = (standard deviation/average)*100. To calculate this coefficient, average values of non-cryopreserved and cryopreserved seeds were considered. The higher the difference between the two materials compared, the higher the overall coefficient of variation.


Moreover, in shoots, the total phenolics and cell wall-linked phenolics contents decreased. The effect of LN on other indicators and plant parts were classified as ¨low¨ (Table 2). The ¨high¨ and ¨medium¨ biochemical effects of cryostorage on S. lycopersicum seeds can be summarized as follows. In roots, lower chlorophyll and phenolics levels, and higher malondialdehyde and other aldehyde contents were measured. In shoots, lower phenolics levels and higher peroxidase enzymatic and specific activities were noted. In leaves, higher peroxidase enzymatic and specific activities, and higher levels of cell wall-linked and total phenolics were measured. DISCUSSION It is well established that any plant species during its life cycle is subjected to environmental stress. Stress alters the normal course of plant growth and development, metabolism, and other physiological processes. Photosynthesis is also subject to this alteration and severely affected under stress conditions. Various abiotic stresses decrease the chlorophyll content in plants (1). The decline in chlorophyll content in plants exposed to stress is believed to be due to inhibition of important enzymes, such as δ-aminolevulinic acid dehydratase and protochlorophyllide reductase associated with chlorophyll biosynthesis (58). We measured chlorophyll a and b content as indicators of stress physiology. Such measurements are used in stress/ pigment photo-oxidation studies to derive the chlorophyll a:b ratio as an indicator of chloroplast pigment stability/acclimation responses to environmental challenges (2, 10, 52). Table 2 showed that in our experiments, chlorophyll pigments (a, b, total) decreased in roots and shoots as a result of seed immersion in LN. Such a decrease was not observed in leaves. In our case, we observed an increase in phenolics content in leaves, which was correlated to the increased activity of enzymes involved in the metabolism of phenolic compounds. Some authors [e.g. (53)] stated that phenolics are generally thought to prevent oxidative damage by scavenging active oxygen species and by breaking the radical chain reactions during lipid peroxidation. These antioxidative effects require the reduced form of phenolics, which act as prooxidants in their oxidized form. The antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agents, donors, singlet oxygen quenchers and metal chelators (53). Other biochemical compounds, such as lipid peroxidation markers (e.g. aldehydes), are involved in biotic (13) and abiotic stress (12, 62, 63). Freezing injury induces the production of free radicals, mainly of reactive oxygen species (6). Free radicals attack the lipid fraction of membranes, resulting in the formation of unstable lipid peroxides. Reactive oxygen species are involved in various aspects of seed physiology (4, 7, 37). Martínez-Montero et al. (40) found that the level of malondialdehyde and others aldehydes in the microsomal fraction was higher in cryopreserved sugarcane callus compared to unfrozen controls, during the first 3 days following LN exposure. Levels of malondialdehyde and of other aldehydes, and peroxidase activity, have been described as being closely connected. Malondialdehyde is one of the primary metabolites of plant response to stress (17). Its accumulation results from peroxidation of cell membrane lipids, and it promotes the formation of other aldehydes (43). It is also well documented that as a result of reactive oxygen species action, besides aldehydes, hydrogen peroxide is formed. Then peroxidase activity is increased (3, 9, 14, 25, 35, 43). Table 2 indicates that in our experiment malondialdehyde and other aldehyde levels and peroxidase specific activities increased in roots and shoots after seed exposure to LN, which concurs with the references mentioned above. In tomato leaves, peroxidase specific activity was significantly higher following seed immersion in LN (Table 2). However, it should be noted that we used the Heath and Packer’s (30) method for recording 418

malondialdehyde and other aldehydes as a tentative measure of the involvement of lipid peroxidation. This procedure is not robust due to assay interference from sugars and non-lipid peroxidation carbonyl groups. Changes in sugar metabolism in seeds during germination could interfere with this assay and confound the interpretation of data. Moreover, the measurement of other aldehydic compounds on the basis of subtracting optical densities at two wavelength absorption peaks is not a definitive measure of aldehydic composition (15). In general, tomato roots were more affected by cryostorage compared with other plant parts (average OCV: 49.32%, Table 2). These results support our previous report on LN exposure of common bean seeds (12). We speculate that S. lycopersicum radicles, being relatively large structures, have a far greater volume of cortical and pith parenchyma cells, which generally are much more vacuolated than meristematic cells. Our hypothesis, therefore, is that damage to such parenchymatous tissue, especially as it is relatively extensive, could be the major event dominating the biochemical disorders induced by seed cryopreservation. It is important to mention that during our experiments, some differences were observed between S. lycopersicum and P. vulgaris (12). Tomato seeds showed lower germination percentages in both treatments compared to common bean seeds, possibly due to the different size and/or structure of seeds. Moreover, with tomato, we used seeds sampled from nondomesticated plants, although plants were grown in a greenhouse after collection in the wild. The resulting heterogeneity of the seed lots may contribute to the lower germination achieved. Seed heterogeneity, defined as the production of different types of seeds by a single individual, appears in many different species of angiosperms (33, 41). Morphological heterogeneity may occur in seed size, shape, and also color (5). Seed heterogeneity may affect physiological properties, being associated with ecological strategies that have evolutionary significance (59), including dormancy (18), germination and longevity behavior (16). Several aspects of seed heterogeneity have been reviewed by Matilla et al. (41). Regarding our experiments with common bean (12) and tomato, the importance of the origin of the seeds employed should be highlighted; they were collected from the wild in the case of tomato and produced commercially in the case of bean (12). Seeds collected from the wild have generally a lower germination percentage because of their heterogeneity, non-optimal physiological state and health status (41). We recorded a positive effect of LN exposure on tomato seed germination at 5 days (Table 1), which was not observed in common bean (12). Immersion of tomato seeds in LN may have helped break seed dormancy, which was not necessary in common bean. Although we have not found any literature on physical dormancy breaking in tomato, it has been documented that LN exposure is one of the classical ways of breaking physical dormancy (23, 24, 39, 47). The potential effect of seed LN exposure on the development of S. lycopersicum seedlings in vivo is being studied in our field experimental station. Acknowledgements: This research was supported by the National Secretary of Superior Education, Science and Technology of Ecuador (SENESCYT); the Polytechnic Agriculture and Cattle Husbandry University of Manabi Manuel Félix López (ESPAM-MFL); and the Bioplant Centre (University of Ciego de Avila, Cuba). We are grateful to Mrs Julia Martínez, Mrs Alitza Iglesias, Mrs. Sandra María Llanes, Mrs. Yudelmis de la Pera and Mr René Ramos for their excellent technical assistance. REFERENCES 1. Ahmad P, Sharma S & Srivastava PS (2007) Hort Sci 34, 114-122. 2. Aragón C, Carvalho L, González J, Escalona M & Amâncio S (2012) Plant Cell Rep 31, 757-769. 419

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