Copper homeostasis influences the circadian clock in Arabidopsis

Abstract

Almost every aspect of plant physiology is influenced by diurnal and seasonal environmental cycles, which suggests that biochemical oscillations must be a pervasive phenomenon in the underlying molecular organization. The circadian clock is entrained by light and temperature cycles and controls a wide variety of endogenous processes that enable plants to anticipate the daily periodicity of environmental conditions. Several previous reports suggest a connection between copper (Cu) homeostasis and the circadian clock in different organisms other than plants. However, the nature of the Cu homeostasis influence on chronobiology remains elusive. Cytosolic Cu content could oscillate since Cu regulates its own transporters expression. We recently reported how the deregulation of Cu homeostasis in Arabidopsis transgenic plants affects the expression of two MYB transcription factors which are nuclear components of the circadian clock. In this addendum, we hypothesize the advantages that could be derived from the influence of metal homeostasis on plant circadian rhythms and their significance.

Tightly regulated Cu homeostasis networks have been traditionally attributed to providing Cu essential cuproproteins and avoiding the harmful effects of excess Cu. Cu homeostasis in the plant model Arabidopsis thaliana has been recently reviewed^1,2 and the cellular responses to Cu deficiency are now starting to be deciphered.³ A family of high affinity Cu transport proteins denoted COPT (COPT1-5) participate in Cu uptake towards the cytosol.⁴ Once in the cytosol, Cu chaperones deliver the metal to target cuproproteins or to heavy metal P-type ATPases (HMA5-8), which either incorporate the metal to cuproproteins located in intracellular compartments or contribute to Cu detoxification when in excess.^1,2,5

In response to Cu deficiency, the Arabidopsis SQUAMOSA promoter binding-like protein SPL7 mediates transcriptional activation through its binding to the reiterative cis regulatory elements (GTAC motifs) present within the promoter of multiple target genes, including those coding for the high affinity Cu transporters COPT1 and COPT2.⁶ On the other hand, SPL7 transcriptional activation is precluded in the presence of elevated intracellular Cu levels.³ The Cu-regulated expression of COPT genes together with COPT-mediated Cu transport activity integrate a negative auto-regulatory feedback loop. By making reasonable assumptions that could cause a sufficient delay in this negative feedback loop, the system could theoretically produce a self-sustained oscillation of cytosolic Cu level by an alternative periodic transfer of Cu from the cytosol to internal stores and back to the cytosol.⁷

Deregutated Copper Homeostasis Alters Circadian Rhythms

In order to test the negative auto-regulatory feedback loop described above, we obtained transgenic plants that constitutively overproduce either COPT1 or COPT3 Cu transporters (C1^OE and C3^OE plants).⁸ Judging by the expression changes of well-known Cu status markers, C1^OE and C3^OE plants display increased endogenous Cu content which is perceived by the Cu transcriptional factor SPL7. Moreover, it is noteworthy that the soil-grown C1^OE and C3^OE plants display certain phenotypes that were not observed when wild-type plants were grown with elevated Cu levels, which may result from deregulated Cu homeostasis; this is perhaps due to the blocking of the auto-regulatory feedback loop and the putative subsequent daily oscillation in cytosolic Cu as the Cu-dependent expression of the transporter was eliminated. Some of these phenotypes included not only delayed flowering time compared to wild type plants under long day conditions that invert under short-day ones, but longer hypocotyls than wild-type seedlings on white light which also invert when grown in the darkness (Fig. 1). Interestingly, both the differential flowering time and hypocotyl length are reminiscent of plants with altered circadian rhythms.^9,10 A common basis for the molecular mechanism of the endogenous circadian clock in eukaryotes seems to consist in transcriptional and post-translational interlocked auto-regulatory feedback loops.¹¹ Two MYB transcription factors: CIRCADIAN AND CLOCK ASSOCIATED (CCA1) and LATE ELONGATED HYP°COTYL (LHY) participate in the core of the Arabidopsis circadian clock. In the presence of excess external Cu, the LHY and CCA1 expression levels decrease in a dose-dependent manner.⁸ Furthermore, different data indicate that the oscillation amplitude of the LHY expression decreases and its phase is delayed by Cu, although the period remains unchanged (Fig. 2).

Figure 1.

Phenotypes associated with altered circadian rhythms in COPT-overexpressing Arabidopsis plants. Flowering time, measured as the number of rosette leaves at the time of floral transition of the plants grown in soil, in long- and short-day photoperiods. Hypocotyl length, measured in 7-day-old seedlings grown at 23°C on MS plates in continuous light or darkness. WT, wild type; COPT^OE, COPT-overexpressing plants.

Figure 2.

Scheme of the luciferase activity circadian oscillation in pLHY:luc seedlings grown on MS plates with low (red line) and high (blue line) copper levels. After entrainment in 12 h light/12 h dark photoperiod and 23°C/16°C temperature cycles, seedlings were subjected to free running conditions with constant light and temperature (23°C).

Is Metal Homeostasis Interdependent on the Circadian Clock?

To explain the influence of Cu on the circadian clock a single GTAC motif, which may account for SPL7-mediated transcriptional regulation,³ is present in both the LHY and CCA1 promoters.⁸ Conversely, several cytosolic Cu influx (COPT) and efflux (HMA) transporters have putative promoter elements that are associated with circadian regulation (Table 1). Furthermore, the SPL7 transcription factor also has a circadian element in its promoter (Table 1). A mutual influence at the transcriptional level could imply an interdependent connection between the circadian clock and Cu homeostasis. Moreover, this interdependence would favour the postulated oscillation in cytosolic Cu derived from the auto-regulatory feedback loop.

Table 1.

Theoretical analysis of the promoters of diverse Cu-homeostasis components

Cu-homeostasis component	MIPS code	GTAC	EE/CCA1	ME/SORLIP 1	TBX/SBX/PBX
COPT1	At5g59030	4	0	0	0
COPT2	At3g46900	4	2	1	1
COPT3	At5g59040	3	4	0	3
COPT5	At5g20650	0	1	0	2
HMA5	At1g63440	0	0	1	2
HMA6 (PAA1)	At4g33520	1	0	2	0
HMA7 (RAN1)	At5g44790	4	4	0	1
HMA8 (PAA2)	At5g21930	0	0	1	0
SPL7	At5g18830	0	0	1	1

The MIPS code and the number of GTAC motifs and elements associated with circadian regulation are indicated. The analyzed promoter regions consisted of 1,000 bp upstream of the translational start site, except for: COPT5 (480 bp), HMA6 (443 bp) and HMA8 (363 bp). The analyzed consensus sequences for the putative circadian elements are: Evening element¹⁵ (EE), AAA TAT CT; CCA1 binding site,¹⁶ AAA AAT CT; Morning element¹⁷ (ME), AAC CAC; Sequence Over-Represented in Light-Induced Promoters¹⁸ (SORLIP 1), GCC AC; Late night-specific telo box (TBX), AAA CCC T; Starch box (SBX), AAG CCC; Protein box (PBX), ATG GGC C.¹⁹

The central circadian oscillator of transcriptional and post-translational feedback loops could be considered an integrator of external environmental cycles as well as some internal rhythmic cytosolic metabolites and other intracellular conditions (Fig. 3) that probably extend oscillations to the entire cell metabolism and its intercellular environment.¹²

Figure 3.

Model of Arabidopsis central oscillator functioning. The core of the circadian oscillator is entrained by environmental rhythmic information from light and temperature cycles, and is synchronized with internal endogenous biochemical oscillators, such as Ca²⁺ metabolism and plausibly Cu homeostasis, to dynamically adapt to the cyclic changes occurring under intracellular compartmentalized conditions, such as redox and stromal pH changes. Putative influences are represented by dotted lines.

Some small cytosolic metabolites displaying rhythmic variations could be considered as input pathways which are rhythmically regulated by feedback from an oscillator and will, thus, contribute to the rhythmic input to that oscillator to thereby increase robustness and inertia, even when the environment is constant. Cyclic adenosine diphosphate ribose (cADPR) is proposed to act as both an output from and input to transcriptional/post-translational feedback loops.¹³ cADPR could act as an upstream regulator of Ca²⁺ oscillation in Arabidopsis (Fig. 3). Interestingly, Ca²⁺ fluxes have been reported to participate as signalling events of the presence of Cu in the medium.¹⁴ Although further work is needed to understand the role of Ca²⁺ in Cu signaling, this fact suggests an additional explanation for the interaction between the Cu homeostasis network and the circadian clock. Other internal daily cycling conditions, such as chloroplastic pH and redox changes, could also be involved in metal homeostasis processes, including compartmentalised protein folding and complexation (Fig. 3). Thus to complete a global dynamic picture of the connection between diurnal rhythms under biochemical intracellular conditions, an essential task is to add a temporal dimension to the study of the transition metal homeostasis networks.

Addendum to: Andrés-Colás N, Perea-García A, Puig S, Peñarrubia L. Deregulated copper transport affects Arabidopsis development especially in the absence of environmental cycles. Plant Physiology. 2010;153:170–184. doi: 10.1104/pp.110.153676.