Paul H. Li

Seasonal alteration of the cytosolic and nuclear Ca ²⁺ concentrations of spruce ( Picea engelmannii Parry) and brome grass ( Bromus inermis Leyss) was investigated by the antimonate precipitation cytochemical technique. Electron microscopic (EM) observations revealed that electron-dense Ca ²⁺ antimonate deposits, an indication of Ca ²⁺ localization, were seen mainly in the vacuole, the cell wall and the intercellular space in samples of both species, collected on 14 July 1997. Few deposits were found in the cytosol and nuclei, showing a low resting level during summer months. On 8 Aug. 1997 following a decrease in daylength of 1 hour and 12 minutes, Ca ²⁺ accumulation was initiated in spruce with increased cytosolic and nuclear Ca ²⁺ deposits, but not in brome grass. On 8 Sept. 1997, Ca ²⁺ accumulation occurred in the cytosol of brome grass. This followed a drop in ambient temperature to 12 °C. Cytosolic and nuclear Ca ²⁺ deposits continued to increase in spruce. Controlled experiments confirmed that it was the low temperature, not shortening daylength, that triggered Ca ²⁺ accumulation in brome grass. High cytosolic and nuclear Ca ²⁺ concentrations lasted about three months in spruce from early August to early November. However, the high cytosolic and nuclear Ca ²⁺ concentrations in brome grass lasted only about 20 days from early September to the end of the month. During winter and spring, both species had low resting cytosolic and nuclear Ca ²⁺ concentrations. The relationship between the duration of the high cytosolic and nuclear Ca ²⁺ concentrations and the status of the developed dormancy/cold hardiness is discussed in light of current findings.

Seasonal alteration of the cytosolic and nuclear Ca²⁺ concentrations of spruce (Picea engelmannii Parry) and brome grass (Bromus inermis Leyss) was investigated by the antimonate precipitation cytochemical technique. Electron microscopic (EM) observations revealed that electron-dense Ca²⁺ antimonate deposits, an indication of Ca²⁺ localization, were seen mainly in the vacuole, the cell wall and the intercellular space in samples of both species, collected on 14 July 1997. Few deposits were found in the cytosol and nuclei, showing a low resting level during summer months. On 8 Aug. 1997 following a decrease in daylength of 1 hour and 12 minutes, Ca²⁺ accumulation was initiated in spruce with increased cytosolic and nuclear Ca²⁺ deposits, but not in brome grass. On 8 Sept. 1997, Ca²⁺ accumulation occurred in the cytosol of brome grass. This followed a drop in ambient temperature to 12 °C. Cytosolic and nuclear Ca²⁺ deposits continued to increase in spruce. Controlled experiments confirmed that it was the low temperature, not shortening daylength, that triggered Ca²⁺ accumulation in brome grass. High cytosolic and nuclear Ca²⁺ concentrations lasted about three months in spruce from early August to early November. However, the high cytosolic and nuclear Ca²⁺ concentrations in brome grass lasted only about 20 days from early September to the end of the month. During winter and spring, both species had low resting cytosolic and nuclear Ca²⁺ concentrations. The relationship between the duration of the high cytosolic and nuclear Ca²⁺ concentrations and the status of the developed dormancy/cold hardiness is discussed in light of current findings.

Previous studies of maize suspension‐cultured cells showed that abscisic acid (ABA) treatment at warm temperatures improved the tolerance of cells to subsequent chilling. In the present study, it is shown that both ABA‐treated and untreated maize cells accumulated proline in response to chilling. However, ABA‐treated cells displayed less lipid peroxidation during chilling, and thus, unlike untreated cells, were able to retain the accumulated proline intracellularly. Proline application experiments indicate that an intracellular proline level higher than 2 µmole (g FW)⁻¹ prior to chilling was needed to meaningfully reduce chilling‐enhanced lipid peroxidation and significantly improve chilling tolerance. The results suggest that total proline accumulation in ABA‐treated as well as untreated cells during chilling was enough to potentially improve chilling tolerance, but proline leakage rendered the control cells unable to benefit from the endogenous synthesis of proline in relation to the alleviation of chilling injury. Proline participated in chilling tolerance improvement in ABA‐treated maize cells, as evidenced by: (1) the inhibition of proline accumulation by l‐methionine‐d, l‐sulphoximine (MSO), an inhibitor of glutamine synthetase, reduced ABA‐improved chilling tolerance, and (2) the addition of glutamine into the medium prevented the MSO‐induced reduction in chilling tolerance. The revised relationship between proline accumulation and membrane stability at cold is discussed in the light of these current findings.

Chilling (4 °C) induced a prolonged high level of intracellular Ca²⁺ (Ca²⁺ overload) and lipid peroxidation in maize (Zea mays L. cv Black Mexican Sweet) cultured cells. However, such Ca²⁺ overload and enhanced lipid peroxidation were not seen in abscisic acid (ABA)‐treated cells, which had an improved chilling tolerance. A Ca²⁺ ionophore, A23187, caused Ca²⁺ overload in both ABA‐treated maize cells and the untreated control, whereas an enhanced lipid peroxidation was detected only in the control. The high level of active oxygen species (AOS) in the control during chilling at 4 °C could be reduced by the presence of lanthanum (La³⁺), a Ca²⁺ channel blocker, in the medium. Moreover, both the A23187‐induced lipid peroxidation and AOS production in the control could be reduced by extracellular EGTA, a Ca²⁺ chelator. Laser‐scanning confocal microscopy revealed that mitochondria were one of the major AOS sources under chilling and during A23187 treatment. In vitro assays showed that superoxide production in isolated maize mitochondria was enhanced by the presence of Ca²⁺. Findings suggest that chilling‐induced Ca²⁺ influx in the control triggers a marked generation of AOS, which in turn results in the enhanced lipid peroxidation. The ability of ABA‐treated cells to avoid the chilling‐induced Ca²⁺ influx may serve as a mechanism that prevents the chilling‐induced oxidative stress and thus results in less chilling injury.

Chilling tolerance was increased in suspension‐cultured cells and seedlings of maize (Zea mays L. cv ‘Black Mexican Sweet’) grown in media containing glycinebetaine (GB). A triphenyl tetrazolium chloride (TTC) reduction test indicated that after a 7 d chilling period at 4 °C, cells treated with 1 mm GB at 26 °C for 1 d had a survival rate (30%) that was twice as high as that of untreated controls. The addition of 2·5 m M GB to the culture medium resulted in maximum chilling tolerance (40%). The results of a cell regrowth assay were consistent with viability determined by the TTC method. In suspension‐cultured cells supplemented with various concentrations of GB, accumulation of GB in the cells was proportional to the GB concentration in the medium and was saturated at a concentration of 240 μmol (g DW)⁻¹. The degree of increased chilling tolerance was positively correlated with the level of GB accumulated in the cells. The increased chilling tolerance was time‐dependent; i.e. it was first observed 3 h after treatment and reached a plateau after 14 h. Feeding seedlings with 2·5 m M GB through the roots also improved their chilling tolerance, as evidenced by the prevention of chlorosis after chilling for 3 d at 4 °C/2 °C. Lipid peroxidation, as expressed by the production of malondialdehyde, was significantly reduced in GB‐treated cells compared with the untreated controls during chilling. These results suggest that increased chilling tolerance may be due, in part, to the reduction of lipid peroxidation of the cell membranes in the presence of GB.

Electron microscopic observations revealed that electron-dense antimonate Ca²⁺ deposits were mostly localized in the vacuole and the intercellular space in both maize and winter wheat when their seedlings were grown at 25°C. The reaction products—cerium phosphate deposits of Ca²⁺- ATPase activity were mainly seen at the cytoplasmic side of the plasma membrane. Few cerium phosphate deposits also were observed on the nuclear envelope. In both species, after 1 or 3 h 2°C chilling, antimonate Ca²⁺ deposits increased in the cytosol and the nucleus, but cerium phosphate deposits showed no visible difference compared to their corresponding 25°C seedlings. After 12, 24, or 72 h chilling, maize seedlings still maintained a high level of antimonate Ca²⁺ deposits in the cytosol and the nucleus. During these periods, maize Ca²⁺-ATPase, as indicated by the number of cerium phosphate deposits, became less and less active as chilling proceeded. In winter wheat, the increased cytosolic and nuclear antimonate Ca²⁺ deposits were restored to a low resting level after 12, 24, or 72 h chilling, while the Ca²⁺-ATPase was maintained active, contrary to maize scenario. The transient cytosolic and nuclear Ca²⁺ increase and the activities of Ca²⁺-ATPase during chilling are discussed in relation to plant chilling injury and cold acclimation.