Accumulation of a clock-regulated transcript during flower-inductive darkness in pharbitis nil

Abstract

To clarify the molecular basis of the photoperiodic induction of flowering in the short-day plant Pharbitis nil cv Violet, we examined changes in the level of mRNA in cotyledons during the flower-inductive photoperiod using the technique of differential display by the polymerase chain reaction. A transcript that accumulated during the inductive dark period was identified and a cDNA corresponding to the transcript, designated PnC401 (P. nil C401), was isolated. RNA-blot hybridization verified that levels of PnC401 mRNA fluctuated with a circadian rhythm, with maxima between 12 and 16 h after the beginning of the dark period) and minima of approximately 0. This oscillation continued even during an extended dark period but was damped under continuous light. Accumulation of PnC401 mRNA was reduced by a brief exposure to red light at the 8th h of the dark period (night-break treatment) or by exposure to far-red light at the end of the light period (end-of-day far-red treatment). These results suggest that fluctuations in levels of PnC401 mRNA are regulated by phytochrome(s) and a circadian clock and that they are associated with photoperiodic events that include induction of flowering.

Figure 1

Preferential accumulation of PnC401 mRNA in dark-treated cotyledons. Seedlings of cv Violet were grown under LL for 6 d after germination of seeds and then transferred to SD conditions (16 h of dark; flower-inductive conditions) or LD conditions (LL; noninductive conditions). Cotyledons were harvested at the 12th and 16th h of each photoperiodic treatment. Total RNA (20 μg per lane) was fractionated by gel electrophoresis and allowed to hybridize to a 3′ fragment of PnC401 cDNA.

Figure 2

DNA gel-blot analysis of the PnC401 gene. Genomic DNA was prepared from cv Violet, digested with EcoRI (E), BamHI (B), and HindIII (H), and subjected to DNA gel-blot hybridization. Full-length PnC401 cDNA was used as the probe. A, Low-stringency conditions. B, High-stringency conditions. Numbers at left indicate mobilities of markers with lengths in kilobases.

Figure 3

Effects of various photoperiodic treatments on the level of PnC401 mRNA in cotyledons. Seedlings of cv Violet were grown under LL for 6 d and then subjected to various photoperiodic treatments: A, DD; B, SD (16 h of darkness); or C, NB (10-min exposure to light at the 8th h of darkness). The cotyledons of 15 plants were harvested every 4 h during photoperiodic treatments and used for extraction of RNA. Total RNA (20 μg per lane) was fractionated by gel electrophoresis and allowed to hybridize to full-length PnC401 cDNA. Uniformity of the loading and transfer of the RNA was confirmed by reprobing the blots with a cDNA fragment for 16S rRNA of P. nil. Relative levels of PnC401 mRNA were calculated from the intensity of each radioactive band, as determined with an imaging plate: D for A, E for B, and F for C.

Figure 4

Effects of exposure to red and FR light on the level of PnC401 mRNA in cotyledons. Seedlings were grown under LL for 6 d and then exposed to 16 h of darkness with or without various kinds of light exposure, as follows: EOD-FR, a 10-min exposure to FR light just before darkness; NB-F, a 5-min exposure to FR light at the 8th h of darkness; NB-R, a 5-min exposure to red light at the 8th h of darkness (NB); and SD, 16th h of darkness. Total RNA (20 μg) was isolated from cotyledons at the 10th and 16th h of each treatment and analyzed. Uniformity of the loading and transfer of the RNA was confirmed by reprobing the blots with the cDNA fragment for 16S rRNA of P. nil.

Figure 5

Effects of a varietal difference in CNL on the level of PnC401 mRNA. CNL of cv Violet is about 10 h and that of cv Kidachi is about 9 h at 25°C. The cotyledons of 15 plants of each cultivar were harvested at indicated times during inductive darkness (SD) or LL (LD). Total RNA (20 μg per lane) was fractionated by gel electrophoresis and allowed to hybridize to full-length PnC401 cDNA. Uniformity of the loading and transfer of the RNA was confirmed by reprobing the blots with cDNA for 16S rRNA of P. nil.

Figure 6

Levels of PnC401 mRNA in different organs. Total RNA was isolated from various organs of seedlings of cv Violet. Lanes: R, root; S, stem; P, petiole; C, cotyledon; and L, first leaf. Organs were harvested at the peak time (at the 16th h of dark treatment) in the circadian oscillation of levels of the transcript in cotyledons. Total RNA (20 μg per lane) was fractionated by gel electrophoresis and allowed to hybridize to full-length PnC401 cDNA.