Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Feb 20;32(4):511-23.
doi: 10.1038/emboj.2012.330. Epub 2012 Dec 14.

Circadian Clock Adjustment to Plant Iron Status Depends on Chloroplast and Phytochrome Function

Affiliations
Free PMC article

Circadian Clock Adjustment to Plant Iron Status Depends on Chloroplast and Phytochrome Function

Patrice A Salomé et al. EMBO J. .
Free PMC article

Abstract

Plant chloroplasts are not only the main cellular location for storage of elemental iron (Fe), but also the main site for Fe, which is incorporated into chlorophyll, haem and the photosynthetic machinery. How plants measure internal Fe levels is unknown. We describe here a new Fe-dependent response, a change in the period of the circadian clock. In Arabidopsis, the period lengthens when Fe becomes limiting, and gradually shortens as external Fe levels increase. Etiolated seedlings or light-grown plants treated with plastid translation inhibitors do not respond to changes in Fe supply, pointing to developed chloroplasts as central hubs for circadian Fe sensing. Phytochrome-deficient mutants maintain a short period even under Fe deficiency, stressing the role of early light signalling in coupling the clock to Fe responses. Further mutant and pharmacological analyses suggest that known players in plastid-to-nucleus signalling do not directly participate in Fe sensing. We propose that the sensor governing circadian Fe responses defines a new retrograde pathway that involves a plastid-encoded protein that depends on phytochromes and the functional state of chloroplasts.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
The period of the circadian clock reflects available Fe concentrations. (A) Fe deficiency induces chlorosis in seedlings. Photographs of Col-2 seedlings grown for 10 days on Hoagland medium containing 0.25, 5 or 20 μM FeHBED. (B, C) Inverse relationship between free-running period length and Fe supply. TOC1:LUC activity (B) and period length (C) of seedlings grown on Hoagland medium with various Fe concentrations. (D, E) Free-running period as a linear function of the log of Fe concentrations. (D) TOC1:LUC period length plotted against log(Fe concentration) follows a linear trend, with R2=0.97. (E) TOC1:LUC and CAB2:LUC period maintains a linear relationship to log(Fe concentration) from 0.25 to 100 μM FeHBED. (F, G) Period lengthening under Zn excess is rescued by increased Fe supply. TOC1:LUC activity (F) and period length (G) for seedlings grown on Hoagland medium containing 5 μM FeHBED and 1 μM (as control) or 30 μM (for excess) ZnSO4, or 50 μM FeHBED and 30 μM ZnSO4. **Significantly different (two-tailed Student's t-test, P<0.001). NS, not significant.
Figure 2
Figure 2
Lagging circadian phase in Fe-deficient seedlings during entrainment. (A) Low Fe supply results in a shift in circadian phase to a later time under entraining regime. TOC1:LUC activity in Col-2 seedlings subjected to light-dark cycles (0–48 h), and later released into constant light (48–96 h). Seedlings were grown on Hoagland medium containing low (1 μM) or high (20 μM) FeHBED. (B) The reporters CCA1:LUC, PRR7:LUC and TOC1:LUC share the same lag in phase under low Fe conditions. **Significantly different between low and high Fe conditions (two-tailed Student's t-test, P≤0.002). (C) The elf3-1 mutant can respond to Fe levels before becoming arrhythmic. TOC1:LUC activity for Col-2 and elf3-1 seedlings during light-dark cycles (24–48 h), and released into constant light (48–72 h).
Figure 3
Figure 3
Free-running period in hy6, a mutant in the chloroplast haem oxygenase HO1, is independent of Fe supply. (A, B) Free-running period remains constant in hy6, regardless of the Fe concentrations in the growth medium. (A) TOC1:LUC activity in Col-2 and hy6 seedlings grown on Hoagland medium containing 1 or 20 μM FeHBED. (B) TOC1:LUC period length in Col-2 and hy6 seedlings grown on Hoagland medium and various Fe concentrations. *,**Significantly different (two-tailed Student's t-test, *P=0.09; **P≤0.002). (C) Circadian phase in hy6 is not affected by Fe concentrations in the growth medium. TOC1:LUC activity in hy6 grown on Hoagland medium containing 1 or 20 μM FeHBED, during light-dark cycles (0–48 h) and in constant light (48–96 h). (D) Chlorosis in hy6 is not due to a defect in Fe uptake. Representative images of 2-week-old seedlings grown on untreated soil (−Fe) or Fe-supplemented soil (+ Fe) soaked with 0.5% (w/v) sequestrene. (E) Increasing Fe supply does not rescue the low chlorophyll content in hy6. Chlorophyll content of Col-0 and hy6 seedlings grown on Hoagland medium with various Fe concentrations. (F) Equivalent responses of Fe status marker FRO2 and FER1 transcript levels in hy6 and Col-0 seedlings. FRO2 and FER1 transcript levels were determined by qPCR and normalized to UBIQUITIN 10 levels (see Materials and methods for details).
Figure 4
Figure 4
Assessment of haem as a potential circadian signalling molecule. (A) Supplementation with high concentrations of haem shortens circadian period of Fe-deficient seedlings. TOC1:LUC activity in Col-2 seedlings grown on Hoagland medium containing 0.25 μM FeHBED only, or supplemented with 5 or 50 μM haem. (B) Haem feeding rescues chlorosis caused by Fe deficiency in Col-0, but not in irt1. Representative 10-day-old seedlings grown on Hoagland medium containing 0.25 μM FeHBED (−Fe), 50 μM hemin (+haem) or 50 μM FeHBED (+Fe). (C) Haem feeding does not shorten TOC1:LUC period in irt1. TOC1:LUC period length in Col-2 and irt1 seedlings grown on Hoagland medium containing 0.25 or 20 μM FeHBED or hemin. **Significantly different (two-tailed Student's t-test, P≤0.01). NS, not significant (P>0.5). (D) Root surface Fe3+ chelate reductase activity responds less strongly to haem than to Fe supplementation. Activity was measured of whole roots of 10-day-old Col-0 seedlings grown on Hoagland medium containing 0.25–20 μM FeHBED (+Fe), or containing 0.25 μM FeHBED and 0.25–50 μM hemin (+haem). (E) The biosynthetic pathway leading from haem to phytochrome chromophore. (F) The new hy2 allele hy2Wisc displays a typical long hypocotyl phenotype. Seven-day-old Col-2 and hy2Wisc seedlings grown on Hoagland medium containing 5 μM FeHBED. (G) hy2Wisc displays a short period under low Fe conditions. TOC1:LUC activity in Col-2 and hy2Wisc seedlings grown on Hoagland medium containing 1 μM FeHBED. (H) hy2Wisc shares the circadian Fe insensitivity of hy6. TOC1:LUC period lengths in Col-2 and hy2Wisc seedlings grown on Hoagland medium with various Fe supply. **Significantly different (two-tailed Student's t-test, P<0.01).
Figure 5
Figure 5
A block in chloroplast protein translation impairs circadian Fe responses. (A) Col-0 seedlings grown on Hoagland medium containing various Fe supply, alone or in combination with 50 μM erythromycin or 10 μg/ml chloramphenicol. (B, C) Circadian period length in seedlings treated with the plastid-specific protein translation inhibitor erythromycin responds less to Fe sufficiency. TOC1:LUC activity (B) and period lengths (C) in Col-2 seedlings grown on Hoagland medium containing various Fe supply and 50 μM erythromycin. (D, E) The plastid and mitochondria protein translation inhibitor chloramphenicol abolishes circadian Fe responses and results in a constitutive long period. TOC1:LUC activity (D) and period lengths (E) in Col-2 seedlings grown on Hoagland medium containing various Fe supply and 10 μg/ml chloramphenicol. **Significantly different between control and treated seedlings (two-tailed Student's t-test, P≤0.02).
Figure 6
Figure 6
Non-circadian and circadian Fe responses are light dependent. (A, B) Hypocotyl elongation is not affected by Fe deficiency. (A) Representative Col-0 and irt1 etiolated seedlings grown in the dark for 7 days on Hoagland medium containing 0.25 or 5 μM FeHBED. (B) Hypocotyl lengths of Col-2 and irt1 etiolated seedlings as a function of Fe supply. (C, D) Expression of FRO2 and FER1 is induced by Fe deficiency (FRO2) or sufficiency (FER1) only in light-grown seedlings (C), but not in etiolated seedlings (D). (E, F) The circadian clock of Col-2 and hy6 etiolated seedlings does not adjust its period to match the Fe supply. TOC1:LUC activity (E) and period lengths (F) in Col-2 and hy6 etiolated seedlings grown on Hoagland medium containing various Fe supply. Period lengths for Col-2 and hy6 are not significantly different (P>0.4).
Figure 7
Figure 7
Coupling of the circadian clock to the circadian Fe sensor depends on phytochromes and HEMERA. (AC) The photoreceptor mutant phyA-211 phyB-9, like hy6, maintains a short FRP even under low Fe conditions. TOC1:LUC activity (A), period lengths (B) and CT phase values (C) of Col-2 and phyA-211 phyB-9 seedlings grown on Hoagland medium containing 1 or 20 μM FeHBED. (D, E) Blocking early phy signalling with a mutant in HEMERA compromises circadian Fe responses. TOC1:LUC activity (D) and period lengths (E) of Col-2 and hmr-2 seedlings grown on Hoagland medium with various Fe supply. **Significantly different between Col-2 and mutant seedlings (two-tailed Student's t-test, P≤0.02). (F) A model for circadian Fe responses in A. thaliana. See text for details. The red arrows represent Fe uptake.

Comment in

Similar articles

See all similar articles

Cited by 28 articles

See all "Cited by" articles

References

    1. Arnon DI (1949) Copper enzymes in isolated chloroplasts. polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1–15 - PMC - PubMed
    1. Chen M, Galvao RM, Li M, Burger B, Bugea J, Bolado J, Chory J (2010) Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes. Cell 141: 1230–1240 - PMC - PubMed
    1. Chory J, Peto CA, Ashbaugh M, Saganich R, Pratt L, Ausubel F (1989) Different roles for phytochrome in etiolated and green plants deduced from characterization of Arabidopsis thaliana mutants. Plant Cell 1: 867–880 - PMC - PubMed
    1. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743 - PubMed
    1. Colangelo EP, Guerinot ML (2004) The essential basic helix-loop-helix protein FIT1 is required for the iron deficiency response. Plant Cell 16: 3400–3412 - PMC - PubMed

Publication types

Feedback