Plastid signals remodel light signaling networks and are essential for efficient chloroplast biogenesis in Arabidopsis

Abstract

Plastid signals are among the most potent regulators of genes that encode proteins active in photosynthesis. Plastid signals help coordinate the expression of the nuclear and chloroplast genomes and the expression of genes with the functional state of the chloroplast. Here, we report the isolation of new cryptochrome1 (cry1) alleles from a screen for Arabidopsis thaliana genomes uncoupled mutants, which have defects in plastid-to-nucleus signaling. We also report genetic experiments showing that a previously unidentified plastid signal converts multiple light signaling pathways that perceive distinct qualities of light from positive to negative regulators of some but not all photosynthesis-associated nuclear genes (PhANGs) and change the fluence rate response of PhANGs. At least part of this remodeling of light signaling networks involves converting HY5, a positive regulator of PhANGs, into a negative regulator of PhANGs. We also observed that mutants with defects in both plastid-to-nucleus and cry1 signaling exhibited severe chlorophyll deficiencies. These data show that the remodeling of light signaling networks by plastid signals is a mechanism that plants use to integrate signals describing the functional and developmental state of plastids with signals describing particular light environments when regulating PhANG expression and performing chloroplast biogenesis.

Figure 1.

Allelism of New gun Mutants and cry1 Mutants. (A) Similar long-hypocotyl phenotypes of new gun mutants and cry1 mutants. Seedlings were grown in 25 μmol·m⁻²·s⁻¹ blue light. Representative seedlings (top) and hypocotyl measurements (bottom) are shown. Hypocotyls were measured in blue light and in the dark for each line shown. Error bars indicate 95% confidence intervals (n > 36). (B) Similar gun phenotypes of new gun mutants and cry1 mutants. Seedlings were grown in 125 μmol·m⁻²·s⁻¹ white light on medium that either contained (+Lin) or lacked (−Lin) lincomycin. RNA was extracted and Lhcb mRNA levels were determined by RNA gel blotting using 3.0 μg of RNA. The levels of Lhcb transcripts were normalized to total RNA stained with methylene blue. Numbers below each lane indicate the amount of hybridized RNA as a percentage of hybridized RNA in untreated wild-type seedlings grown in the same light condition. (C) Rough mapping of the long hypocotyl in blue light phenotype. The wild-type hypocotyl phenotype was rough-mapped based on an analysis of 118 chromosomes from F2 progeny that were obtained from a cry1-401 (Col-0) × Ler cross and exhibited wild-type hypocotyl lengths in 25 μmol·m⁻²·s⁻¹ blue light. Chromosomes were analyzed using two simple sequence length polymorphism (SSLP) markers, nda22 and smd1 (see Supplemental Table 2 online). BAC clones that contain the SSLP marker sequences for nda22 and smd1 are indicated. The numbers of centromere proximal recombinants (top) and centromere distal recombinants (bottom) identified with each marker are indicated. (D) CRY1 nucleotide and derived amino acid substitutions found in the new gun mutants. The altered codons and the resulting amino acid substitutions found in new cry1 mutants are indicated. Lines and boxes indicate introns and exons, respectively. The photolyase-related (PHR) domain and the DAS motifs have been reviewed by Lin and Shalitin (2003).

Figure 2.

Expression of Lhcb and Rbcs in gun1 and cry Mutants after Chloroplast Biogenesis Was Blocked. Seedlings were grown in either 50 μmol·m⁻²·s⁻¹ blue or 125 μmol·m⁻²·s⁻¹ white light on medium that either contained (+Lin) or lacked (−Lin) lincomycin. The levels of Lhcb and Rbcs mRNA were quantitated as described for Figure 1B, except that 4.0 μg of RNA was used.

Figure 3.

Expression of Lhcb and Rbcs in gun1 and cry1 after Chloroplast Biogenesis Was Blocked with Various Inhibitors of Chloroplast Biogenesis. Seedlings were grown in 50 μmol·m⁻²·s⁻¹ blue light on medium that lacked any inhibitor of chloroplast biogenesis (−Lin) or contained lincomycin (+Lin), erythromycin (+Ert), or norflurazon (+Nfl). Lhcb and Rbcs mRNA levels were quantitated as described for Figure 1B, except that 4.0 μg of RNA was used.

Figure 4.

Expression of Lhcb and Rbcs in cop1-4 and hy5 Mutants after Chloroplast Biogenesis Was Blocked. (A) gun phenotypes of cop1-4. Seedlings were grown in 50 μmol·m⁻²·s⁻¹ blue light on medium that contained (+Lin) or lacked (−Lin) lincomycin. Lhcb and Rbcs mRNA levels were quantitated as described for Figure 1B. (B) gun phenotypes of hy5. The growth of seedlings and the quantitation of the levels of Lhcb and Rbcs mRNA were as described for (A).

Figure 5.

Expression of Lhcb and Rbcs in the Dark or in Various Qualities of Light. (A) Lhcb and Rbcs expression levels in darkness, white light, blue light, red light, and far-red light. Seedlings were grown on medium containing lincomycin in the dark or in 125 μmol·m⁻²·s⁻¹ white light, 25 μmol·m⁻²·s⁻¹ blue light, 35 μmol·m⁻²·s⁻¹ red light, or 2 μmol·m⁻²·s⁻¹ far-red light. The levels of Lhcb and Rbcs mRNA were quantitated as described for Figure 1B, except that 2.5 μg of RNA was used. (B) Expression of Lhcb and Rbcs in white light and in a combination of blue and red light. Seedlings were grown on medium containing (+Lin) or lacking (−Lin) lincomycin in either white light (W) or a combination of blue and red light (B+R). Fluence rates were as described for (A). The levels of Lhcb and Rbcs mRNA were quantitated as described for (A). The numbers below the B+R (−Lin) lanes indicate the amount of hybridized RNA as a percentage of mRNA in the untreated control grown in white light (−Lin).

Figure 6.

Expression of Lhcb and Rbcs in gun1, phyA, and phyB Mutants after Chloroplast Biogenesis Was Blocked. Seedlings were grown in 125 μmol·m⁻²·s⁻¹ white light on medium that lacked (−Lin) or contained (+Lin) lincomycin. The levels of Lhcb and Rbcs mRNA were quantitated as described for Figure 1B.

Figure 7.

Effects of Plastid Development on the Fluence Rate Response of Lhcb and Rbcs. Seedlings were grown in white light fluence rates of 0, 1.0, 10, 50, or 125 μmol·m⁻²·s⁻¹ in either the presence (+Lin) or absence (−Lin) of lincomycin. The levels of Lhcb and Rbcs mRNA were quantitated as described for Figure 1B. For the untreated wild type, the number below each lane indicates the amount of hybridized RNA as a percentage of hybridized RNA in the untreated wild type grown in 125 μmol·m⁻²·s⁻¹ white light.

Figure 8.

Chlorophyll-Deficient Cotyledons in gun1 and gun1 cry Mutants. (A) Percentage of seedlings exhibiting chlorophyll-deficient phenotypes. Seedlings were grown on medium without an inhibitor of chloroplast biogenesis. The total number of seedlings and the seedlings that were visibly chlorophyll-deficient were counted after 6 d of growth in 125 μmol·m⁻²·s⁻¹ white light. Four independent experiments were performed, and each experiment contained a total of ∼50 seedlings. Error bars represent 95% confidence intervals between independent experiments. (B) Chlorophyll-deficient seedlings. Representative wild-type (Col-0) and chlorophyll-deficient mutant seedlings are shown after 6 d (Col-0, gun1-101, gun1-1 cry1, and gun1-1 cry1 cry2) or 7 d (gun1-1 and gun1-1 cry1) of growth in white light. Arrows indicate chlorophyll-deficient areas. Bars = 2 mm.

Figure 9.

HL Sensitivity of gun1 and Light Signaling Mutants. (A) gun1 and light signaling mutant seedlings grown in the indicated fluence rates of continuous white light. One-day-old etiolated seedlings were irradiated with the indicated fluence rates of continuous white light for 6 d. Representative seedlings are shown. Bars = 2 mm. (B) Comparisons of total chlorophyll levels in gun1 and light signaling mutants in various fluence rates of continuous white light. Seedlings were grown as described for (A). Chlorophyll was extracted from at least three samples for each line in each condition. Error bars represent 95% confidence intervals.

Figure 10.

Model for PhANG Regulation by a Network of Plastid and Light Signaling Pathways. The current model for the GUN1-dependent plastid-to-nucleus signaling pathway was adapted from Koussevitzky et al. (2007). In this model, a second messenger (indicated with Y) that requires GUN1 for either its production or transduction (dotted arrows) triggers a plastid-to-nucleus signaling pathway that represses PhANGs. A plastid signal(s) that is independent of GUN1 (indicated with X) represses both Lhcb and Rbcs in the dark (data not shown) and also converts cry1 and one or more photoreceptors that perceive red light into negative regulators of Lhcb and Rbcs. cry1 becomes a negative regulator of Lhcb when X converts HY5 from a positive to a negative regulator of Lhcb. Under these same conditions, Rbcs is induced by cry1 and simultaneously repressed by a combination of blue and red light.