HSP90 Contributes to Entrainment of the Arabidopsis Circadian Clock via the Morning Loop

Abstract

The plant circadian clock allows the synchronization of internal physiological responses to match the predicted environment. HSP90.2 is a molecular chaperone that has been previously described as required for the proper functioning of the Arabidopsis oscillator under both ambient and warm temperatures. Here, we have characterized the circadian phenotype of the hsp90.2-3 mutant. As previously reported using pharmacological or RNA interference inhibitors of HSP90 function, we found that hsp90.2-3 lengthens the circadian period and that the observed period lengthening was more exaggerated in warm-cold-entrained seedlings. However, we observed no role for the previously identified interactors of HSP90.2, GIGANTEA and ZEITLUPPE, in HSP90-mediated period lengthening. We constructed phase-response curves (PRCs) in response to warmth pulses to identify the entry point of HSP90.2 to the oscillator. These PRCs revealed that hsp90.2-3 has a circadian defect within the morning. Analysis of the cca1, lhy, prr9, and prr7 mutants revealed a role for CCA1, LHY, and PRR7, but not PRR9, in HSP90.2 action to the circadian oscillator. Overall, we define a potential pathway for how HSP90.2 can entrain the Arabidopsis circadian oscillator.

Keywords: Arabidopsis; HSP90; circadian clock.

Figure 1

hsp90.2-3 has a circadian period phenotype. Free-running profile of CCR2::LUC in Ws-2 and hsp90.2-3 seedlings under constant red–blue light (LL) after plants were prior trained to 12:12 cycles of (A and C) light–dark (LD) or (B and D) warm–cold (WC). Plants were released into free-running conditions at (zeitgeber time) ZT36. (C and D) Mean period estimates of (A) and (B), respectively. Error bars represent SEM. *** P < 0.001. Significance determined using a Student’s t-test. In each experiment, 48 WT and hsp90.2-3 seedlings were analyzed for rhythms under each entrainment protocol. All experiments repeated at least once. CPS, counts per second.

Figure 2

Geldanamycin (GDA) treatment has an additive effect on hsp90.2-3 circadian period phenotype. Period estimates of the free-running period (FRP) of CCR2::LUC in the (A) Ws-2 or (B) hsp90.2-3 seedlings treated with either DMSO or with 2 µM GDA under constant red–blue light (LL). For (A), plants were entrained under 12:12 light–dark cycles. Plants in (B) were entrained under the stated entrainment conditions. For both (A) and (B), GDA was applied upon transfer to free-running conditions. Error bars represent SEM. ** P < 0.01 and *** P < 0.001. Significance determined via a Student’s t-test. In each experiment, the FRP of 48 seedlings was examined. Each experiment was repeated at least once.

Figure 3

Geldanamycin (GDA) lengthening of circadian period is not dependent on GI or ZTL. Period estimates of the free-running profile of (A) gi-11 CCR2::LUC or (B) ztl-21 CAB2::LUC. Plants were entrained under 12:12 cycles of light–dark (LD) or warm–cold (WC) before transfer to free-running conditions. Next, 2 µM of GDA was applied upon transfer to free-running conditions. Error bars represent SEM. ** P < 0.01 and *** P < 0.001. Significance determined using a Student’s t-test. In each experiment, 48 seedlings of WT and the respective mutant were examined under each entrainment condition, apart from gi-11 LD (GDA) where n = 30. All experiments were repeated at least once.

Figure 4

hsp90.2-3 has a morning phase defect. Ws-2 and hsp90.2-3 CCR2::LUC plants were entrained for 7 days under 12/12 light–dark cycles before being exposed to 3-hr long pulses of 27°. Phase-response curves were then constructed by plotting the observed phase shift in CCR2::LUC expression against the circadian time that heat pulses were administered. Positive values represent phase advances and negative values represent phase delays. *** P < 0.001; n.s., no significant difference. Significance determined via a Student’s t-test. Error bars represent pooled SE.

Figure 5

Geldanamycin (GDA) fails to lengthen circadian period in the cca1 or lhy mutant. (A and B) Period estimates of CCR2::LUC profile under free-running conditions in Ws-2 [wild-type (WT)], cca1-11, and lhy-21 mutants treated with or without 2 µM GDA. Plants were prior entrained to light–dark (A) or warm–cold (B) cycles before being released into free-running conditions. GDA treatment was applied upon transfer to free-running conditions. Error bars represent SEM. In each experiment, 48 WT and mutant seedlings were examined under each entrainment condition. n.s., no significance; *** P < 0.001. Significance determined by a Student’s t-test. All experiments were repeated at least once.

Figure 6

The effect of geldanamycin (GDA) treatment on period length is disrupted in the prr7 and prr7/prr9 background. Period estimates of the free-running profile of CCA1::LUC in the Col-0, prr7-3, prr9-1, and prr7-3/prr9-1 background. Plants were entrained under (A) light–dark or (B) warm–cold cycles before being released into free-running conditions. Plants were treated with or without 2 µM GDA upon transfer to free-running conditions. Error bars represent SEM. ** P < 0.01 and *** P < 0.001; n.s., no significance. Significance determined via a Student’s t-test. In each experiment, 48 WT and mutant seedlings were examined under each entrainment condition. All experiments were repeated at least once.

Figure 7

An expanding role of HSP90 within the Arabidopsis circadian oscillator. HSP90 has been previously shown to interact with ZTL to regulate both periodicity and, under heat stress, the stability of the oscillator. GI and HSP90 are thought to cooperatively stabilize ZTL activity. Here, we have found that Hsp90 also signals independently of GI and ZTL through the morning loop components CCA1/LHY and PRR7. We did not detect a direct effect of HSP90 on regulating CCA1/LHY expression, and HSP90 was also found previously to not regulate PRR7 expression. Therefore, this indicates that HSP90 is signaling via an as yet unidentified protein to regulate CCA1/LHY and PRR7 activity. Purple lines indicate an interaction (direct or indirect), red lines indicate a repressive interaction, and blue lines highlight the effect of the interaction on the oscillator.