Search

The circadian pacemaker in plants is a hierarchical multioscillator system that directs and maintains a 24‐hr oscillation required for organism homeostasis and environmental fitness. Molecular clockwork within individual tissues and organs acts cell autonomously, showing differences in circadian expression of core oscillators and their target genes; there are functional dominance and coupling in the complex regulatory network. However, molecular characteristics of organ‐specific clocks are still unknown. Here, we showed the detached shoot and root possess dynamic circadian protein–protein interactions between clock core components, periodicity in organs exhibits a difference. The period length difference between shoot and root was not remarkable in prr7‐3 and prr7‐3 prr9‐1 mutants. In addition, the phase transition curve indicated that shoot and root clock respond differently to the resetting cues of ambient temperature. PRR9 and PRR7 compensate circadian period between 22°C and 28°C in shoot, not in root. The circadian rhythms of PRR9 or PRR7 transcript accumulation showed no difference at 22°C and 28°C in shoot, but differences were observed in root. In summary, our results reveal the specificity of dynamic circadian protein–protein interactions in organ‐autonomous clocks and the critical roles of PRR9 and PRR7 in mechanisms regulating temperature compensation in aerial shoot system. We found the difference of circadian period length of dynamic circadian protein–protein interactions between shoot and root. PRR9 and PRR7 were found to contribute significantly to organ‐specific circadian periodicity maintenance and in the temperature compensation of aerial shoot clock. To the best of our knowledge, this is the first example in plants monitoring clock protein interactions in real time in individual organs. Having demonstrated the uniqueness and consistency of clock components function in aerial shoot and subterranean root, we are able to pose a variety of intriguing questions about the adaptive evolution and hierarchical coupling in the plant clock system. [ABSTRACT FROM AUTHOR]

Melatonin is a well‐studied neurohormone oscillating in a 24‐h cycle in vertebrates. Phytomelatonin is widespread in plant kingdom, but it remains elusive whether this newly characterized putative hormone underlies the regulation by daily rhythms. Here, we report phytomelatonin signaling, as reflected by changes in endogenous concentrations of phytomelatonin and expression of genes associated with biosynthesis of phytomelatonin (AtSNAT1, AtCOMT1, and AtASMT) and its receptor (AtPMTR1), shows 24‐h oscillations in Arabidopsis. The variation of reactive oxygen species (ROS) production and scavenging and expression of ROS‐related genes significantly decrease in pmtr1 and snat and increase in PMTR1‐OE seedlings, indicating the rhythmicity in phytomelatonin signaling is required for maintenance of ROS dynamics. Additionally, the ROS signaling feedback influences the expression of AtSNAT1, AtCOMT1, AtASMT, and AtPMTR1, suggesting the phytomelatonin and ROS signaling are coordinately interrelated. The pmtr1 mutant plants lose diurnal stomatal closure, with stomata remaining open during daytime as well as nighttime and mutants showing more water loss and drought sensitivity when compared with the wild‐type Col‐0 plants. Taken together, our results suggest that PMTR1‐regulated ROS signaling peaks in the afternoon and may transmit the darkness signals to trigger stomatal closure, which might be essential for high water‐use efficiency and drought tolerance. [ABSTRACT FROM AUTHOR]

Bioluminescence resonance energy transfer (BRET) is a natural biophysical phenomenon that underlies an emerging technique to monitor protein–protein interactions in living cells in real time. Here, we present a series of technical advances to enhance the utility of the BRET assay in plants. A series of recombination cloning vectors was generated to accelerate the expression of proteins tagged with Renilla luciferase or yellow fluorescent protein under transient assay conditions and in stable transgenic plants. Working in stably transformed Arabidopsis or tobacco, we then detected BRET between three pairs of candidate interaction partners: dimerization of the E3 ubiquitin ligase COP1, interaction between COP1 and the B-box protein STH, and interaction between the light regulatory bZip transcription factors HY5 and HYH. A codon-optimized version of the Renilla luciferase gene resulted in improved expression in Arabidopsis. Renilla luciferase was active in a variety of subcellular organelles, including plastids, mitochondria, peroxisomes and Golgi stacks. In a survey of the Arabidopsis light signaling machinery as a model system, we estimated the likelihood that a known protein–protein interaction can be documented using BRET. Finally, we show that Renilla luciferase may serve as a reporter of protein stability in a cycloheximide chase assay. [ABSTRACT FROM AUTHOR]