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Monitoring characteristics of cold hardiness for perennial ryegrass organs from the view point of dynamic states of water

Mari Iwaya-Inoue1, Rina Matsui2 and Masataka Fukuyama1

1Laboratory of Crop Science, Department of Plant Resources, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka
812-8581, Japan. Email,
Department of Plant Resources, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki,
Fukuoka 812-8581, Japan


Dynamic states of water in the leaves and roots of perennial ryegrass (Lolium perenne L.) exposed to cold stress were studied by using 1H-NMR. NMR spin-lattice relaxation times (T1) of leaves in Arrhenius plots linearly decreased with temperature but increased as temperature decreased below 0oC. However, spin-spin relaxation times (T2) of the leaves increased as temperature decreased from 20 to -20oC. The T2 value of the long fraction (associated with vacuole) in leaves decreased to about 600s at -25oC, but that of the short fraction was about 10s, and the relative value of signal intensity of the long fraction decreased to about 0.2 at -25oC. The T2 values of the two fractions in roots decreased to about 1ms at -10oC. Judging from T2, electrolyte leakage and TTC reduction tests, both vacuolar and cytoplasmic compartments of leaves and roots froze at these temperatures. From these results, analysis of Arrhenius plots of T1 and T2 is a sensitive and non-invasive method to evaluate primary response of perennial ryegrass organs to the cold stress.

Media summary

Primary response to cold hardiness for leaves and roods in perennial ryegrass was determined by Arrhenius plots of 1H-NMR relaxation times (T1 , T2 ).

Key Words

Arrhenius plots, Cold stress, NMR relaxation times (T1, T2), Supercooling ability, Water compartment


Perennial ryegrass (Lolium perenne L.) has been widely cultivated as a pasture in cool temperate regions and it is important to select cold-tolerant perennial ryegrass genotypes for adaptation to northern climates (Ebdon et al. 2002). The over-wintering ability was mainly determined by the concentration of nonstructural carbohydrates in the roots rather than in the tops in four temperate perennial grasses (Tamura and Moriyama 2001). Although the mechanism of thermo-tolerance in plants has been extensively studied, little is known about dynamic states of water which affect cellular metabolism. Since the mobility and characteristic of cell-associated water is closely related to the condition of plant tissues (Ishida et al. 2000), NMR relaxation time, such as spin-lattice relaxation time (T1) and spin-spin relaxation time (T2) of water protons have been applied to the studies of higher plant tissues exposed to thermal stresses (Kaku et al. 1985; Iwaya-Inoue et al. 1993; Maheswari et al. 1999). In chilling-injury studies, Arrhenius plots of a variety of biological reaction rates have been examined, and non-linear temperature dependency or the presence of discontinuity has been seen as a diagnostic phase transition of membrane lipids and other parameters (Iwaya-Inoue et al. 1989; 2004b; Schreiber 2001). In this study, we have characterized motional modes of tissue water in perennial ryegrass to determine cold stress sensitivities of organs such as leaves and roots by using 1H-NMR spectroscopy.


Leaves and roots for perennial ryegrass (Lolium perenne L. cv. Friend) grown with a 12h/12h day/night cycle at 15oC for 3 to 4 weeks were used for following experiments. T1 and T2 were measured by using a 1H-NMR spectrometer with a magnet operating at 25MHz for 1H (M25A, JEOL Ltd.). For chilling treatments, the temperature of the samples was lowered in steps of 5oC from 20 to -15oC (or -25oC).

For T1 measurements, the saturation recovery method (90-τ-90 pulse sequence) was used. T2 was measured by the Carr-Purcell-Meiboom-Gill (CPMG) method with (90ox -τ-180oy -2τ-180oy-2τ…) pulse sequence. The solid-echo (90o-τ-90o) method was also applied for measurement of T2 below 1ms. A decay curve of echo signal was analyzed by using a non-linear least-square method on semi-log plots of signal intensity. For detailed analysis, two component analysis was carried out (Iwaya-Inoue et al. 2004c).

Figure 1.Arrhenius plots of T1 values (A) and T2 values (B) of long and short fractions in leaves of perennial ryegrass cv. Friend exposed to a decrease in temperature from 20 to -25oC. Closed symbols(●) indicate T1 values (A) or T2 values (B) of long fractions and open symbols (○) indicate those of short fractions in leaves. Arrows indicate cooling process

Results and Discussion

Water in plant tissues can be characterized by different proton relaxation times (Isobe et al. 1999; Iwaya-Inoue and Nonami 2003). T1 values of long and short fractions in leaves were constant or linearly decreased as temperature was lowered from 20 to 0oC (Fig. 1A). T1 values of seedlings in chilling-insensitive pea plants (Pisum sativum L.) also decreased linearly as the temperature decreased from 20 to 0oC (Iwaya-Inoue et al. 1989). On the contrary, T1 of the two components in ryegrass leaves gradually increased as the temperature decreased from 0 to -15oC. T1 values of Vigna radiata hypocotyls have been reported to show a reversible gradual increase after the tissues were exposed to 0oC for 1h (Iwaya-Inoue et al. 1993). It seems likely that the gradual prolongation of T1 in plant tissues exposed to chilling stress depends partly on changes in cytoplasmic pH (Yoshida et al. 1989).

A difference in T2 values of biological tissues can also be interpreted in terms of the differences of the ratio of “free water” to “bound water” (Walter et al. 1989). T2 values of the two fractions in the leaves were constant at temperatures between 20 and 0oC (Fig. 1 B). T2 s of sugar solutions depend less on temperature but T1 linearly decreased as the temperature decreased from 30 to 0oC (Iwaya-Inoue et al. 2004a). Thus, it was assumed that the suppression of water mobility determined by T2 was more intensified at higher temperatures. T2 in the long fraction indicating over 200 ms is thought to be mainly associated with vacuole and the fraction with the short T2 less than 60ms is thought to be associated with the cytosol and apoplastic region in the leaves. In the temperature range from 0 to -20oC, gradual increasing of the T2 value of the long fraction increased slightly, but that of the short fraction decreased linearly. T2 values of the long fractions and those of the short fractions were less than 1ms and 30s, respectively at -25oC. The T2 value below 100s is thought to be associated with water tightly bound to macromolecules in cells (Hills and Remigereau 1997). They stated that a peak of T2 corresponding to the vacuole in parenchyma tissue of apple, vanished at -3oC showing that the vacuolar compartment had frozen, but the two peaks corresponding to the cytoplasm and cell wall compartments were observed indicating that these compartments remain unfrozen. These results indicate that vacuolar water in ryegrass leaf tissues disappeared at -25oC. Additionally, T2 values of both long and short fractions in roots were constant at temperatures between 20 and 0oC, and T2 value of both fractions decreased slightly at temperatures ranging from 0 to -10oC (data not shown). An abrupt decrease in T2 value of the two components in both leaves and roots was accompanied with the decrease in signal intensity of the long fraction.

Judging from the ion leakage and TTC reduction (data not shown), these organs were severely damaged after the exposure to these temperatures. The shortening of T2 has been attributed to a decline in membrane permeability under freezing injury in wheat crowns (Chen and Gusta 1978). Therefore, the abrupt decrease in T2 reflected the intracellular freezing of tissue water in leaves between -20 and -25oC, but in roots at-10oC in the perennial ryegrass cultivar ‘Friend’. From these results, a drastic decrease of the relative value of signal intensity as well as that of T2 values in the two water components reflected the freezing of vacuoles and cytoplasmic water in the plant organ cells.


Arrhenius plots of both T1 and T2 suggested that dynamic states of water reflected the cold-tolerance of the leaves and roots. Although there is a little information on the supercooling ability of herbaceous plants, T2 determination revealed that perennial ryegrass ‘Friend’ has supercooling ability at -20 to -25oC in leaves and at -10oC in roots. In general, plant roots are protected from temperature stresses more efficiently than the other organs, because the soil temperature is more stable than the air temperature as an environmental factor.

These results show the importance of selecting temperature resistant perennial ryegrass organs for adaptation to cool climate. Arrhenius plots of NMR relaxation times (T1, T2) in the organs of perennial ryegrass cultivar provided a sensitive and non-invasive way for evaluating tissue response to cold stress.


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