Biochemical Responses of chsl chs2 and chs3 Mutants to Chilling

Class 1 chilling-sensitive mutants include chsl, chs2 . and chs3 . In addition to previous observations regarding the responsiveness of chsl plants to chilling (Hugly et al. 1990; Patterson et al. 1993 ; Schneider et al. 1995b), we hereby show that upon transfer of 2-week-old plants from a normal temperature of 22°C to a moderate chill ing temperature of 10°C all class 1 chs mutants, including chsl, chs2. and chs3. turned yellow, wilted and eventually died (Fig. 7.1). The yellowing and growth arrest phenotypes become visible in 7 days after transfer to chilling temperatures, and these phenotypes become more severe as the time of exposure to chilling increased up to 3 weeks (Fig. 7.1). Furthermore, we found that in addition to what has been reported previously for chsl (Hugly et al. 1990), chs3 mutants are also extremely sensitive to low temperatures, as they showed a severe dwarfism phenotype when grown at a moderate chilling temperature of 18°C (Fig. 7.2). Overall, all class 1 chilling-sensitive mutants are very sensitive to chilling, and turn yellow and wilt in just a few days after exposure to low temperatures.

To further evaluate the effects of chilling on chsl, chs2. and chs3 plants, we examined their chlorophyll content, photosynthesis efficacy and electrolyte leakage rates at various periods after exposure to 10°C. It can be seen that the earliest event that was observed within 4 days of exposure to chilling was a sharp decline in chlorophyll content (Fig. 7.3a), and this was followed by a gradual decrease in photosynthetic efficacy (determined by measuring chlorophyll fluorescence; Fv/Fm ratio) which continued up to 21 days of exposure to 10°C (Fig. 7.3b). An increase in electrolyte leakage rates was evident after 7 days at 10°C and gradually became more severe as the plants were kept for longer periods at 10°C (Fig. 7.3c).

In accordance with the observed decreases in chlorophyll contents and photosynthetic efficacy, we found that chsl, chs2. and chs3 plants were unable and did not accumulate starch in their leaves as observed in WT plants (Fig. 7.4). Following exposure to low temperatures, WT plants continued to produce assimilates by photosynthesis, but since growth was slowed down they accumulated starch. In contrast, the chsl, chs2, and chs3 mutants were defected in their photosynthetic machinery (as observed by the decrease in chlorophyll content and photosynthesis efficacy) and, therefore, did not produce sugars and did not accumulate starch following exposure to low temperatures (Fig. 7.4).

Fig. 7.1 Phenotypes of wild-type and chsl, chs2. and chs3 mutants following transfer to chilling conditions. Plants were grown for 2 weeks at 22°C and afterwards transferred to a chilling temperature of 10°C

wt chsl chs2 chs3

Fig. 7.2 Effects of a moderate growth temperature of18°Con the phenotypes of wild-type and chsl, chs2, and chs3 mutants. Plants were grown from sowing at 22 or 18°C, and photographs were taken after 3 weeks of growth at each temperature

Fig. 7.3 Effects of chilling on chlorophyll content, photosynthesis efficacy, and electrolyte leakage rates of wildtype and chsl, chs2, and chs3 mutants. Plants were grown for 2 weeks at 22°C and afterwards transferred to a chilling temperature of 10°C. (a) Chlorophyll content, (b) photosynthesis efficacy, and (c), electrolyte leakage. Data are means ± S.E. of five replications

Fig. 7.3 Effects of chilling on chlorophyll content, photosynthesis efficacy, and electrolyte leakage rates of wildtype and chsl, chs2, and chs3 mutants. Plants were grown for 2 weeks at 22°C and afterwards transferred to a chilling temperature of 10°C. (a) Chlorophyll content, (b) photosynthesis efficacy, and (c), electrolyte leakage. Data are means ± S.E. of five replications

Another response of chsl, chs2, and chs3 mutants to chilling was enhanced accumulation of ROS in general, and H2O2 particularly, as observed by DAB staining experiments (Fig. 7.5). It can be seen that WT plants remained clear and did not accumulate H2 O2 following exposure to chilling, whereas leaves of chsl, chs2, and chs3 mutants were stained by brown spots indicating presence of H2O2 (Fig. 7.5). The accumulation of H2O2 was somewhat more pronounced in chs2 and chs3 plants as compared with chsl, and after exposure to the lower temperature of 4°C as compared with 13°C (Fig. 7.5).

Starch accumulation (iodine staining) 22°C 13°C 4°C

wt c/is2

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