Cysteine, gamma-Glutamylcysteine, and Glutathione Levels in Maize Seedlings : Distribution and Translocation in Normal and Cadmium-Exposed Plants

The levels of cysteine (Cys), γ-glutamylcysteine (γEC), and glutathione (GSH) were measured in the endosperms, scutella, roots, and shoots of maize (Zea mays L.) seedlings. GSH was the major thiol in roots, shoots, and scutella, Cys predominated in endosperms. The endosperm, scutellum, and functional phloem translocation were required for maintenance of GSH pools in roots and shoots of 6-day-old seedlings. Exposure of roots to 3 micromolar Cd, besides causing a decline in GSH, caused an accumulation of γEC, as if the activity of GSH synthetase was reduced in vivo. [³⁵S]Cys injected into endosperms of seedlings was partly metabolized to [³⁵S]sulfate. The scutella absorbed both [³⁵S]sulfate and [³⁵S]Cys and transformed 68 to 87% of the radioactivity into [³⁵S]GSH. [³⁵S]GSH was translocated to roots and shoots in proportion to the tissue fresh weight. Taken together, the data supported the hypothesis that Cys from the endosperm is absorbed by the scutellum and used to synthesize GSH for transfer through the phloem to the root and shoot. The estimated flux of GSH to the roots was 35 to 60 nanomoles per gram per hour, which totally accounted for the small gain in GSH in roots between days 6 and 7. For Cd-treated roots the GSH influx was similar, yet the GSH pool did not recover to control levels within 24 hours. The estimated flux of GSH to the entire shoot was like that to the roots; however, it was low (11-13 nanomoles per gram per hour) to the first leaf and high (76-135 nanomoles per gram per hour) to the second and younger leaves.     

Abscisic Acid induces anaerobiosis tolerance in corn

Flooding is a frequently occurring environmental stress that can severely affect plant growth. This study shows that treatment of corn (Zea mays L.) seedlings with abscisic acid (ABA) increases their tolerance to anoxia 10-fold over untreated seedlings and twofold over seedlings treated with water. Corn seedlings stressed anoxically for 1 day showed only 8% survival when planted in vermiculite. Pretreatment of root tips with 100 micromolar ABA or water for 24 hours before the 1 day anoxic stress increased the anoxic survivability of seedlings to 87% and 47%, respectively. Cycloheximide (5 milligrams per liter), added together with ABA, reduced the seedling survival rate, indicating that the induction of anoxic tolerance in corn by ABA was partly a result of the synthesis of new proteins. ABA treatment induced a threefold increase in alcohol dehydrogenase enzyme activity in corn roots. However, after 24 h of anoxia, alcohol dehydrogenase enzyme activity between the ABA-pretreated and non-pretreated corn roots was not significantly different. The results indicated that ABA played an important role in inducing anoxic tolerance in corn and that the induced tolerance was probably mediated by an increase in alcohol dehydrogenase enzyme activity before the anoxic stress.     

Hydroxylation of N-(Delta-Isopentenyl)adenine to Zeatin: Relative Activities of Organ Systems from Actinidia arguta

By incubating explants from Actinidia arguta seedlings on a nutrient medium supplemented with 20 to 30 micromolar N⁶-(Δ²-isopentenyl)adenine (i⁶Ade) and then measuring zeatin (io⁶Ade) accumulation in tissues, the distribution of i⁶Ade hydroxylase activities in whole plants could be determined. Based on analyses with three entire plants, it is estimated that, as an organ system, roots contain approximately 68% of the plant's hydroxylase, while stems and leaves account for about 26% and 6%, respectively, of the total activity. Depending on the part of the root examined, hydroxylase activities ranged from 20 to 148 nanomoles io⁶Ade accumulated per gram fresh weight per 24 hours of incubation. Stem activities ranged from 17 to 165 nanomoles per gram fresh weight per 24 hours with the lowest activities being found at the tip. Leaf activities were substantially lower (1-10 nanomoles per leaf depending on position) than either root or stem.     

Measurement of the pyrophosphate content of plant tissues

Pyrophosphate (PPi) was measured in pea (Pisum sativum L.) and corn (Zea mays L.) tissues by using an enzymic method based on PPi-dependent phosphofructokinase (PPi-PFK). Different organs of pea and corn seedlings were extracted to determine if PPi is present in sufficient amounts to serve as a substrate for the PPi-PFK activity in these tissues. The amount of PPi is at least 14% to 70% that of the ATP content in shoots and roots of peas and corn; and, for various plant tissues, ranges from 5 to 39 nanomoles of PPi per gram fresh tissue weight. We conclude that PPi is available as a substrate for the glycolytic function of PPi-PFK in plants. Furthermore, the presence of substrate amounts of PPi in plant tissues implies that plant energetics also must be evaluated in terms of PPi as an energy source and phosphate donor.     

Turnover of dhurrin in green sorghum seedlings

The turnover of dhurrin in green seedlings of Sorghum bicolor (Linn) Moench var Redland x Greenleaf, Sudan 70 has been investigated using glyphosate and pulse-labeling studies with ¹⁴C-tyrosine and [¹⁴C]shikimic acid. The rate of dhurrin breakdown was 4.8 nanomoles per hour in the shoot and 1.4 nanomoles per hour in the root. The rate of dhurrin accumulation in the shoot of 4- to 5-day-old seedlings was high but decreased with age until at the peak period of dhurrin accumulation, the rates of dhurrin synthesis and breakdown were equal. Using a first order equation (an approximation) the rate of dhurrin synthesis (which equals accumulation plus breakdown rates) was 17.4 nanomoles per hour in the shoot and 4.1 nanomoles per hour in the root. In both tissues, the breakdown rate was between 27 and 34% of their synthetic capacity within the experimental period. Dhurrin synthesis in green sorghum seedlings occurred in both the light and dark photoperiods but was faster in the dark period. The result is discussed in relation to the possible metabolic roles of the turnover.     

Development of water conducting capacity in the root systems of young plants of corn and some other c4 grasses

Development of the primary and early nodal roots was studied in Zea mays L., Zea mexicana (Schrad.) Reeves & Mangelsd., Sorghum bicolor (L.) Moench., and Sorghum sudanese (Piper) Stapf. in relation to shoot development. In all the types studied all roots reached lengths of about 30 centimeters before the late metaxylem (LMX) was open, and young plants with total root lengths of around 100 centimeters had almost no open LMX. On average, corn seedlings with up to 36 square centimeters of leaf had no open LMX. The name “immature apices” is suggested for such long but not fully functional roots. In plants up to 50 days old a fairly constant proportion of less than half the total root length had open LMX. A pilot study of stomatal resistance on days of high evaporative demand suggested that young seedlings may show higher resistance than older plants in the afternoon. Estimates of longitudinal permeability of corn roots with only early metaxylem vessels open indicate very steep gradients of water potential would develop under such conditions.     

ATPase in lipid body membranes of castor bean endosperm

Lipid body membranes purified from castor seed endosperm of dry seeds and 4 d old seedlings were found to have an ATPase activity associated with them. This was confirmed by equilibrium density centrifugation of the membranes using acid lipase as a marker enzyme. The specific activity ranged from 45 to 200 nanomoles per milligram protein per minute. The pH optimum was 9.0 but at pH 7.5 nearly 40% of the maximum activity was retained. The apparent K_m for Mg-ATP was 0.5 millimolar. A divalent cation was required for activity and Mg²⁺ was the most effective. Other nucleoside triphosphates were also hydrolyzed but there was no hydrolysis of pyrophosphate or p-nitrophenylphosphate. The ATPase was not inhibited by oligomycin, vanadate, dicyclohexylcarbodiimide, or molybdate but was inhibited by sodium azide. Washing the membranes with increasing concentrations of NaCl removed up to 60% of the ATPase activity but none was removed by 3 millimolar ethylene-diaminetetraacetate.     

Characterization of Fatty Acid biosynthesis in isolated pea root plastids

Fatty acid biosynthesis from Na[1-¹⁴C]acetate was characterized in plastids isolated from primary roots of 7-day-old germinating pea (Pisum sativum L.) seeds. Fatty acid synthesis was maximum at 82 nanomoles per hour per milligram protein in the presence of 200 micromolar acetate, 0.5 millimolar each of NADH, NADPH, and coenzyme A, 6 millimolar each of ATP and MgCl₂, 1 millimolar each of MnCl₂ and glycerol-3-phosphate, 15 millimolar KHCO₃, 0.31 molar sucrose, and 0.1 molar Bis-Tris-propane, pH 8.0, incubated at 35°C. At the standard incubation temperature of 25°C, fatty acid synthesis was essentially linear for up to 6 hours with 80 to 120 micrograms per milliliter plastid protein. ATP and coenzyme A were absolute requirements, whereas divalent cations, potassium bicarbonate, and reduced nucleotides all variously improved activity two- to 10-fold. Mg²⁺ and NADH were the preferred cation and nucleotide, respectively. Glycerol-3-phosphate had little effect, whereas dithiothreitol and detergents generally inhibited the incorporation of [¹⁴C]acetate into fatty acids. On the average, the principal radioactive products of fatty acid biosynthesis were approximately 39% palmitic, 9% stearic, and 52% oleic acid. The proportions of these fatty acids synthesized depended on the experimental conditions.     

Control by ethylene of arginine decarboxylase activity in pea seedlings and its implication for hormonal regulation of plant growth

Activity of arginine decarboxylase in etiolated pea seedlings appears 24 hours after seed imbibition, reaches its highest level on the 4th day, and levels off until the 7th day. This activity was found in the apical and subapical tissue of the roots and shoots where intensive DNA synthesis occurs. Exposure of the seedlings to ethylene greatly reduced the specific activity of this enzyme. The inhibition was observed within 30 min of the hormone application, and maximal effect—90% inhibition—after 18 hours. Ethylene at physiological concentrations affected the enzyme activity; 50% inhibitory rate was recorded at 0.12 microliters per liter ethylene and maximal response at 1.2 microliters per liter. Ethylene provoked a 5-fold increase in the K_m^app of arginine decarboxylase for its substrate and reduced the V_max^app by 10-fold. However, the enzyme recovered from the inhibition and regained control activity 7 hours after transferral of the seedlings to ethylene-free atmosphere. Reducing the endogenous level of ethylene in the tissue by hypobaric pressure, or by exposure to light, as well as interfering with ethylene action by treatment with silver thiosulfate or 2,5-norbornadiene, caused a gradual increase in the specific activity of arginine decarboxylase in the apical tissue of the etiolated seedlings. On the basis of these findings, the possible control of arginine decarboxylase activity by endogenous ethylene, and its implication for the hormone effect on plant growth, are discussed.     

Alteration of thiol pools in roots and shoots of maize seedlings exposed to cadmium : adaptation and developmental cost

Roots of intact 5-day-old maize (Zea mays L.) seedlings were exposed to 3 micromolar Cd during a 7-day period. Cysteine, γ-glutamylcysteine, glutathione (GSH), and Cd-induced acid-soluble thiols (ASTs), including phytochelatins, were quantified in roots and shoots. Adaptation to Cd and its cost to seedling development were evaluated by measuring Cd content, tissue fresh weight, and rate of root elongation. Roots contained 60 to 67% of the Cd in the seedlings between 4 and 7 days of exposure. Exposure to Cd decreased the fresh weight gain in roots from day 4 onward without affecting the shoots. Between days 1.5 and 3.5 of Cd treatment, roots elongated more slowly than controls; however, their growth rate recovered thereafter and exceeded that of controls. Exposure to Cd did not appreciably affect the concentration of cysteine in the seedlings. However, the initial low concentration of γ-glutamylcysteine increased (after a lag of 6 hours in roots and 2 days in shoots), reaching a plateau by day 6 at 28.5 nanomoles per gram of fresh weight in roots and by day 5 at 19.1 nanomoles per gram of fresh weight in shoots. During the first 9 hours of Cd exposure, the concentration of GSH in roots decreased dramatically (at 31.6 nanomoles per gram of fresh weight per hour) and thereafter decreased more slowly than in controls. The depletion of GSH in the roots (366 nanomoles per gram of fresh weight) matched the synthesis of ASTs (349 nanomoles per gram of fresh weight) during the first 48 hours. The concentration of ASTs in roots increased steadily thereafter to reach 662.2 nanomoles per gram of fresh weight by 6 days of Cd exposure. In shoots, Cd had little influence on the concentration of GSH, but ASTs still accumulated to 173.3 nanomoles per gram fresh weight after 5 days. The molar ratio of thiols in ASTs to Cd increased to a maximum of 10.24 in roots after 4 hours and of 4.25 in shoots after 2 days of Cd exposure. After 4 days, the ratio reached a plateau of approximately 2 in roots and between 2 and 3 in shoots, as if a steady state of Cd chelation had been achieved in both organs. The plateau coincided with recovered root elongation or an adaptation to Cd. The reduced fresh weight gain of the roots during this time, however, indicated that the synthesis of Cd-induced thiols was at a cost to root development.     

Stimulation of Root Elongation and Curvature by Calcium

Ca²⁺ has been proposed to mediate inhibition of root elongation. However, exogenous Ca²⁺ at 10 or 20 millimolar, applied directly to the root cap, significantly stimulated root elongation in pea (Pisum sativum L.) and corn (Zea mays L.) seedlings. Furthermore, Ca²⁺ at 1 to 20 millimolar, applied unilaterally to the caps of Alaska pea roots, caused root curvature away from the Ca²⁺ source, which was caused by an acceleration of elongation growth on the convex side (Ca²⁺ side) of the roots. Roots of an agravitropic pea mutant, ageotropum, responded to a greater extent. Roots of Merit and Silver Queen corn also responded to Ca²⁺ in similar ways but required a higher Ca²⁺ concentration than that of pea roots. Roots of all other cultivars tested (additional four cultivars of pea and one of corn) curved away from the unilateral Ca²⁺ source as well. The Ca²⁺-stimulated curvature was substantially enhanced by light. A Ca²⁺ ionophore, A23187, at 20 micromolar or abscisic acid at 0.1 to 100 micromolar partially substituted for the light effect and enhanced the Ca²⁺-stimulated curvature in the dark. Unilateral application of Ca²⁺ to the elongation zone of intact roots or to the cut end of detipped roots caused either no curvature or very slight curvature toward the Ca²⁺. Thus, Ca²⁺ action on root elongation differs depending on its site of application. The stimulatory action of Ca²⁺ may involve an elevation of cytoplasmic Ca²⁺ in root cap cells and may participate in root tropisms.     

Ethylene Evolution from Maize (Zea mays L.) Seedling Roots and Shoots in Response to Mechanical Impedance

The effect of mechanical impedance on ethylene evolution and growth of preemergent maize (Zea mays L.) seedlings was investigated by pressurizing the growth medium in triaxial cells in a controlled environment. Pressure increased the bulk density of the medium and thus the resistance to growth. The elongation of maize primary roots and preemergent shoots was severely hindered by applied pressures as low as 10 kilopascals. Following a steep decline in elongation at low pressures, both shoots and roots responded to additional pressure in a linear manner, but shoots were more severely affected than roots at higher pressures. Radial expansion was promoted in both organs by mechanical impedance. Primary roots typically became thinner during the experimental period when grown unimpeded. In contrast, pressures as low as 25 kilopascals caused a 25% increase in root tip diameter. Shoots showed a slight enhancement of radial expansion; however, in contrast to roots, the shoots increased in diameter even when growing unimpeded. Such morphological changes were not evident until at least 3 hours after initiation of treatment. All levels of applied pressure promoted ethylene evolution as early as 1 hour after application of pressure. After 1 hour, ethylene evolution rates had increased 10, 32, 70, and 255% at 25, 50, 75, and 100 kilopascals respectively, and continued to increase linearly for at least 10 hours. When intact corn seedlings were subjected to a series of hourly cycles of pressure, followed by relaxation, ethylene production rates increased or decreased rapidly, illustrating tight coupling between mechanical impedance and tissue response. Seedlings exposed to 1 microliter of ethylene per liter showed symptoms similar to those shown by plants grown under mechanical impedance. Root diameter increased 5 times as much as the shoot diameter. Pretreatment with 10 micromolar aminoethoxyvinyl glycine plus 1 micromolar silver thiosulfate maintained ethylene production rates of impeded seedlings at basal levels and restored shoot and root extension to 84 and 90% of unimpeded values, respectively. Our results support the hypothesis that ethylene plays a pivotal role in the regulation of plant tissue response to mechanical impedance.     

Quantitation of Adsorption of Rhizobia in Low Numbers to Small Legume Roots

Bacteria adsorbed in low numbers to alfalfa or clover root surfaces were counted after incubation of seedlings in mineral solution with very dilute inocula (less than 10⁵ bacteria per ml) of an antibiotic-resistant strain under defined conditions. After specified washing, bacteria which remained adsorbed to roots were selectively quantitated by culturing the roots embedded in yeast extract-mannitol-antibiotic agar and counting the microcolonies along the root surface; the range was from about 1 bacterium per root (estimated as the most probable number) to 50 bacteria per cm of root length (by direct counting). This simple procedure can be used with any pair of small-rooted plant and antibiotic-resistant bacterium, requires bacterial concentrations comparable to those frequently found in soils, and yields macroscopic localization and distribution data for adsorbed bacteria over the root surface. The number of adsorbed bacteria was proportional to the size of the inoculum. One of every four Rhizobium meliloti cells adsorbed in very low numbers to alfalfa roots resulted in the formation of a nodule. Overall adsorption of various symbiotic and nonsymbiotic bacterial strains to alfalfa and clover roots did not reflect the specificities of these legumes for their respective microsymbionts, R. meliloti and R. trifolii.     

Growth of Carrot and Tomato from Oxamyl-coated Seed and Control of Meloidogyne hapla

Oxamyl was coated on carrot (Daucus carota L. cv. Spartan Fancy-80) and tomato (Lycopersicon esculentum Mill. cv. Glamour) seeds with a polymer sticker for the control of Meloidogyne hapla. The sticker diluted in water 1:1 delayed carrot seedling emergence. Oxamyl at 40 mg/ml in a 1:5 dilution of sticker lowered the rate of carrot seedling emergence until day 13 and plant growth until day 28. Oxamyl at 20 or 40 mg/ml in a 1:5 dilution of sticker on carrot seeds planted in M. hapla-infested muck soil resulted in fewer galled tap roots and fewer galls per root system 4 weeks after planting. Tap root lengths were greater than those of the control. Tomato seedling emergence was delayed and top and root weights were reduced, relative to the control, at 25 days by the sticker diluted 1:1 to 1:3. Oxamyl at 20 or 40 mg/ml in a 1:5 diluted sticker delayed tomato seedling emergence. Top weights of tomato seedlings from seeds coated with 20 mg/ml of oxamyl in a 1:5 diluted sticker planted in a silt loam were greater than control top weights at 4 and 6 weeks. Root weights were greater than those of the control only at 4 weeks. There were fewer galls per gram of root on seedlings from oxamyl-coated seeds and fewer juveniles per pot of soil, relative to the controls, only at 4 weeks.     

Changes in Levels of Intermediates of the C(4) Cycle and Reductive Pentose Phosphate Pathway under Various Light Intensities in Maize Leaves

The rate of CO₂ assimilation and levels of metabolites of the C₄ cycle and reductive pentose phosphate pathway in attached leaves of maize (Zea mays L.) were measured over a range of light intensity from 0 to 1,900 microEinsteins per square meter per second under a saturated CO₂ concentration of 350 microliters per liter and a limiting CO₂ concentration of 133 microliters per liter. The level of ribulose 1,5-bisphosphate (RuBP) stayed almost constant (around 60 nanomoles per milligram chlorophyll [Chl]) from low to high light intensities under 350 microliters per liter. Levels of 3-phosphoglycerate (PGA) increased from 100 to 650 nanomoles per milligram Chl under 350 microliters per liter CO₂ with increasing light intensity. The calculated RuBP concentration of 6 millimolar (corresponded to 60 nanomoles per milligram Chl) was about two times above the estimated RuBP binding-site concentration on ribulose bisphosphate carboxylase-oxygenase (Rubisco) of ~2.6 millimolar in maize bundle sheath chloroplasts in the light. The ratio of RuBP/PGA increased with decreasing light intensity under 350 microliters per liter CO₂. These results suggest that RuBP carboxylation is under control of light intensity possibly due to a limited supply of CO₂ to Rubisco through the C₄ cycle whose activity is highly dependent on light intensity. Pyruvate level increased with increasing light intensity as long as photosynthesis rate increased. A positive relationship between levels of PGA and those of pyruvate during steady-state photosynthesis under various conditions suggests that an elevated concentration of PGA increases the carbon input into the C₄ cycle through the conversion of PGA to PEP and consequently the level of total intermediates of the C₄ cycle can be raised to mediate higher photosynthesis rate.     

Effect of Ethylene Treatment on Polar IAA Transport, Net IAA Uptake and Specific Binding of N-1-Naphthylphthalamic Acid in Tissues and Microsomes Isolated from Etiolated Pea Epicotyls

The effect of ethylene treatment on polar indole-3-acetic acid (IAA) transport, net IAA uptake in the presence and absence of N-1-naphthylphthalamic acid (NPA) and [³H]NPA binding characteristics was investigated in tissue segments or microsomes isolated from etiolated pea (Pisum sativum L. cv Alaska) epicotyls. Basipetal IAA transport in 5 millimeter segments isolated from ethylene-treated seedlings was inhibited by ethylene in a dose-dependent manner. Threshold, half-maximal and saturating concentrations of ethylene were 0.01, 0.55, 10.0 microliters per liter, respectively. This inhibition became apparent after 6 to 8 hours of ethylene treatment. Transport velocity in both control and ethylene-treated tissues was estimated to be 5 millimeters per hour. Net IAA uptake was stimulated in ethylene-treated tissues and the relative ability of the phytotropin NPA to enhance net IAA uptake was reduced in treated tissues. Specific binding of [³H]NPA to microsomes prepared from both control and ethylene-treated tissues was saturable and consistent with the existence of a single class of binding sites with an apparent affinity (K_d) toward NPA of 8 to 9 nanomolar. The density of these binding sites (per milligram protein) was lower (36% of control) in ethylene-treated tissues. Direct application of ethylene to microsomal preparations isolated from untreated seedlings had no effect on the level of specific [³H]NPA binding.     

Distribution of a Take-All Suppressive Strain of Pseudomonas fluorescens on Seminal Roots of Winter Wheat

An antibiotic-resistant strain of Pseudomonas fluorescens, that suppresses take-all of wheat, was used to study the distribution of the bacteria on seminal roots of wheat after being introduced onto seeds. Cells of P. fluorescens were isolated from the entire length of the root, and density of the introduced bacteria declined with the distance from the base of the root. Maximum populations of 10⁵ to 10⁶ CFU and 10³ to 10⁵ CFU per cm of root were detected on sections of roots near the seed and root tip, respectively. The introduced bacteria competed well with indigenous bacteria, comprising at least 25% of the fluorescent pseudomonads detected by plate counts for 48 days after planting.     

Nitrate Reduction in Response to CO(2)-Limited Photosynthesis : Relationship to Carbohydrate Supply and Nitrate Reductase Activity in Maize Seedlings

The effects of CO₂-limited photosynthesis on ¹⁵NO₃⁻ uptake and reduction by maize (Zea mays, DeKalb XL-45) seedlings were examined in relation to concurrent effects of CO₂ stress on carbohydrate levels and in vitro nitrate reductase activities. During a 10-hour period in CO₂-depleted air (30 microliters of CO₂/ per liter), cumulative ¹⁵NO₃⁻ uptake and reduction were restricted 22 and 82%, respectively, relative to control seedlings exposed to ambient air containing 450 microliters of CO₂ per liter. The comparable values for roots of decapitated maize seedlings, the shoots of which had previously been subjected to CO₂ stress, were 30 and 42%. The results demonstrate that reduction of entering nitrate by roots as well as shoots was regulated by concurrent photosynthesis. Although in vitro nitrate reductase activity of both tissues declined by 60% during a 10-hour period of CO₂ stress, the remaining activity was greatly in excess of that required to catalyze the measured rate of ¹⁵NO₃⁻ reduction. Root respiration and soluble carbohydrate levels in root tissue were also decreased by CO₂ stress. Collectively, the results indicate that nitrate uptake and reduction were regulated by the supply of energy and carbon skeletons required to support these processes, rather than by the potential enzymatic capacity to catalyze nitrate reduction, as measured by in vitro nitrate reductase activity.     

Global Changes in DNA Methylation in Seeds and Seedlings of Pyrus communis after Seed Desiccation and Storage

The effects of storage and deep desiccation on structural changes of DNA in orthodox seeds are poorly characterized. In this study we analyzed the 5-methylcytosine (m⁵C) global content of DNA isolated from seeds of common pear (Pyrus communis L.) that had been subjected to extreme desiccation, and the seedlings derived from these seeds. Germination and seedling emergence tests were applied to determine seed viability after their desiccation. In parallel, analysis of the global content of m⁵C in dried seeds and DNA of seedlings obtained from such seeds was performed with a 2D TLC method. Desiccation of fresh seeds to 5.3% moisture content (mc) resulted in a slight reduction of DNA methylation, whereas severe desiccation down to 2–3% mc increased DNA methylation. Strong desiccation of seeds resulted in the subsequent generation of seedlings of shorter height. A 1-year period of seed storage induced a significant increase in the level of DNA methylation in seeds. It is possible that alterations in the m⁵C content of DNA in strongly desiccated pear seeds reflect a reaction of desiccation-tolerant (orthodox) seeds to severe desiccation. Epigenetic changes were observed not only in severely desiccated seeds but also in 3-month old seedlings obtained from these seeds. With regard to seed storage practices, epigenetic assessment could be used by gene banks for early detection of structural changes in the DNA of stored seeds.