Responses of 8-tubulin gene expression to low-temperature exposure (4'C) have been investigated in leaves of Arabidopsis tbaliana. During low-temperature exposure, the patterns of both a-and fl-tubulin isoforms are altered; the effect is smaller for the a-tubulins than for the 8-tubulins, however. An examination of 8-tubulin gene expression revealed that during low-temperature exposure, transcript levels of TUBZ, TUB3, TUB6, and TUBB decrease, whereas those of TUB4, TUB5, and TUB7 remain constant, and the TUB9 transcript level increases. The changes in transcript levels of TUB6, TUB8, and TUB9 were detedable after 6 h of low-temperature treatment. As shown by transcription-blocking experiments, the in vivo decay rates at 25'C are comparable to those at 4°C for TUBS, TUB6, and TU68 mRNAs, whereas TUB9 mRNA appears to be more stable at 4'C than at 25'C. Thus, decreases in transcript levels of TUB6 and TUBB in response to low temperature appear to be regulated at the transcriptional level, and the increase in TUB9 transcript level that results from lowering the temperature from 25'C to 4°C may be due in part to its slower rate of decay at 4'C. When a chimeric gene containing 1061 base pairs of TUB8 5' flanking DNA fused to the &glucuronidase coding region was used to produce transgenic Arabidopsis plants, the chimeric gene expression was down-regulated in response to low temperature as assayed by histochemical localization and RNA gel blots. These results confirm that the alteration of transcript levels of TUB8 in response to low temperature is regulated at the transcriptional level.
Cold‐acclimated stems of red‐osier dogwood (Cornus sericea L.) were sampled in midwinter and early spring and subjected to the following low temperature treatments: (a)0 →−40 → 0°C; (b) 0 →−40 →− 196 → 0°C; (c) 0 →−40 →−196 →−269 →−196 → 0°C; (d) 0 →−40 →−269 →−196 → 0°C; (e) 0 →−196 → 0°C; (f) 0 →−269 →−196 →0°C. The cortical parenchyma cells of the outer stem layers survived exposure to −269°C when pre‐frozen to −40°C and either transferred directly to −269°C or to −196°C and then to −269°C (treatments c and d). Acclimated stems transferred to a greenhouse (22°C) 2 weeks prior to the low temperature treatments deacclimated and were not able to survive freezing to −10°C. Cortical cells of stem samples taken in March, near the time when dogwood naturally deacclimates, survived −196°C (treatment b), but not −269°C (treatment cord). Thus, the freezing tolerance of dogwood varies seasonally from near −10°C to below −269°C.
Cold‐induced depolymerization of cortical microtubules were examined in suspension culture cells of corn (Zea mays L. cv Black Mexican Sweet) at various stages of chilling. In an attempt to determine whether microtubule depolymerization contributes to chilling injury, experiments were carried out with and without abscisic acid (ABA) pretreatment, since ABA reduces the severity of chilling injury in these cells. Microtubule depolymerization was detectable after 1 h at 4°C and became more extensive as the chilling was prolonged. There was little chilling injury after 1 d at 4°C in either ABA‐treated or non‐ABA‐treated cells. After 3 d at 4°C, there was about 26% injury for ABA‐treated and 40% injury for non‐ABA‐treated cells, as evaluated by 2,3,5‐triphenyl‐tetrazolium chloride reduction and by regrowth. After 1d at 4°C, less than 10% of cells retained full arrays of microtubules in both ABA‐treated and non‐ABA‐treated cells, the remainder having either partial arrays or no microtubules. After 3d at 4°C, about 90% of cells showed complete or almost complete depolymerization of microtubules in both ABA‐treated and non‐ABA‐treated cells. ABA did not stabilize the cortical microtubules against cold‐induced depolymerization. In about 66% of ABA‐treated cells and 57% of non‐ABA‐treated cells that had been held at 4°C for 3d, repolymerization of cortical microtubules occurred after 3h at 28°C. These results argue against the hypothesis that depolymerization of cortical microtubules is a primary cause of chilling injury.
We have used double fluorescence labelling to investigate the effect of freezing on microtubules and microfilaments in root‐tip cells of rye (Secale cereale L. cv Rymin). Freezing to ‐5°C (which does not kill these cells) caused partial depolymerization of both, but microfilaments were more resistant than microtubules. When microtubules were stabilized against freeze‐induced depolymerization by pre‐treating seedlings with taxol, microfilaments exhibited enhanced stability as well. Almost all the frozen cells containing taxol‐stabilized microtubules also contained microfilaments. When seedlings were treated with the microtubule‐destabilizing drug APM prior to freezing, microfilaments became more susceptible to freeze‐induced depolymerization than in controls. These data suggest a physical interaction between microtubules and microfilaments in these cells.
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