Shoot Removal Induces Chloroplast Development in Roots via Cytokinin Signaling

Abstract

The development of plant chloroplasts is regulated by various developmental, environmental, and hormonal cues. In Arabidopsis (Arabidopsis thaliana), chloroplast development is repressed in roots via auxin signaling. However, roots develop chloroplasts when they are detached from the shoot. In contrast to auxin, cytokinin positively affects chloroplast development in roots, but the role and signaling pathway of cytokinin in the root greening response remain unclear. To understand the regulatory pathways of chloroplast development in the plant stress response, we examined the mechanisms underlying the conditional greening of detached roots. In wild-type Arabidopsis roots, shoot removal activates type B ARABIDOPSIS RESPONSE REGULATOR (ARR)-mediated cytokinin signaling and induces chlorophyll accumulation and photosynthetic remodeling. ARR1 and ARR12 are essential for up-regulating nucleus- and plastid-encoded genes associated with chloroplast development in detached roots. In this process, WOUND INDUCED DEDIFFERENTIATION1 and class B GATA transcription factors (B-GATAs) act upstream and downstream of ARRs, respectively. Overexpression of B-GATAs promotes root greening, as does shoot removal, dependent on a light signaling transcription factor, LONG HYPOCOTYL5. Auxin represses the root greening response independent of ARR signaling. GNC-LIKE (GNL), a B-GATA, is strongly up-regulated in detached roots via ARR1 and ARR12 but is repressed by auxin, so GNL may function at the point of convergence of cytokinin and auxin signaling in the root greening response.

Figure 1.

Cytokinin signaling functions in the greening of detached roots downstream of wounding signaling. A, GFP fluorescence in roots of the TCSn::GFP line and the wild-type control. B, Chl content in intact and detached roots of the wild type (WT) and mutants involved in cytokinin signaling. C, GFP fluorescence in roots of the TCS::GFP lines with the wild-type and WIND1-SRDX background. D, Chl content in intact and detached roots of the wild type and two independent lines of WIND1-SRDX plants. In A to D, for the detached root sample, roots were excised from 14-d-old seedlings and grown for 7 d, whereas for the intact root control, the shoots of 21-d-old seedlings were removed just before observation. E, Chl content in intact roots of the XVE-WIND1 line. Root Chl content is shown for 21-d-old seedlings treated with 17β-estradiol for 24 h compared with the untreated control (0 h). Student’s t test: *, P < 0.05. For B, D, and E, data are means ± se from three or more independent experiments. Different letters indicate significant differences by the Tukey-Kramer multiple comparison test (P < 0.05). Asterisks indicate significant differences from the wild type of each condition (*, P < 0.05; **, P < 0.01; and ***, P < 0.001, Student’s t test after a Bonferroni correction for multiple comparisons).

Figure 2.

Expression of genes associated with chloroplast differentiation in intact and detached roots. Quantitative RT-PCR analysis of mRNA expression is shown for genes associated with photosynthesis (A), nuclear transcription (B), plastid transcription (C), mitochondrial and peroxisomal functions (D), and root tissues (E) in roots of the wild type and the arr1 arr12 double mutant. Data are means ± se fold difference from the untreated wild type after normalizing to ACTIN8 (n > 3). The sample preparation was as in Figure 1. Different letters indicate significant differences for each gene (P < 0.05, Tukey-Kramer multiple comparison test).

Figure 3.

Comparison of photosystems in detached and intact roots and in leaves. A, Transient fluorescence induction kinetics of Chl in the absence (−) or presence (+) of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Data are means from three independent experiments. Two inflections, J and I, are seen between the levels O (origin) and P (peak) only in the −DCMU samples. B, The 77K Chl fluorescence spectra in 2 μg mL⁻¹ Chl fractions. Representative spectra from three independent experiments are shown. Data were normalized to the emission peak from PSII at 682 nm with background correction at 800 nm.

Figure 4.

Improvement of photosynthetic efficiency in detached roots via cytokinin signaling. Photosynthetic parameters are shown for 21-d-old detached roots and intact roots treated with 6-benzyladenine (BA) of the wild type (A) and the arr1 arr12 double mutant (B) compared with untreated intact roots. In A, wild-type leaves were used as a reference of the photosynthetically competent organ. Data are means ± se (n > 4). F_v/F_m, Maximum quantum yield of PSII; F_v′/F_m′, maximum quantum yield of PSII under light conditions; qP, coefficient of photochemical quenching; Y_NPQ, quantum yield of regulated energy dissipation; Y_NO, quantum yield of nonregulated energy dissipation. Slow induction kinetics under actinic light (intensity, 420 µmol photons m⁻² s⁻¹) are shown for Y_II, and other data were obtained after actinic light exposure for 15 min. Different letters indicate significant differences (P < 0.05, Tukey-Kramer multiple comparison test).

Figure 5.

Involvement of transcription factors in the root greening response after shoot removal. A and B, Chl content in intact and detached roots of each mutant line (A) and intact roots with GNC and GNL overexpression (GNCox and GNLox, respectively) in the wild type (WT) and hy5 mutant background (B). Data are means ± se from three or more independent experiments. n.d., Not determined. C and D, Size (C) and number (D) of green plastids in primary root cells. The horizontal line represents the median; the top and bottom of boxes represent the upper and lower quartiles, respectively; and whiskers represent the range (C, n > 900; D, n > 80). ***, P < 0.001 by Student’s t test. E and F, Expression of photosynthesis-associated genes in intact and detached roots of the gnc gnl double mutant (E) and intact roots of GNCox and GNLox (F). Data are means ± se fold difference from the intact wild-type root samples after normalizing to ACTIN8 (n = 3). G, Plastid DNA content in intact and detached root cells. The content of plastid DNA relative to nuclear DNA was determined by real-time PCR-based quantification of DNA content with rpoB-specific (plastid-encoded) and ACTIN8-specific (nucleus-encoded) primers. Data are means ± se fold difference from intact wild-type root samples (n = 3). Samples at 21 d old were used for all experiments. Different letters indicate significant differences by the Tukey-Kramer multiple comparison test (P < 0.05). Asterisks indicate significant differences from the wild type of each condition (*, P < 0.05; **, P < 0.01; and ***, P < 0.001, Student’s t test after a Bonferroni correction for multiple comparisons).

Figure 6.

Involvement of transcription factors in root photosynthesis in response to shoot removal. A to D, Y_II in intact (A and D), detached (B), and BA-treated intact (C) roots of each line. E to H, Photosynthetic parameters in intact roots of overexpression lines of each transcription factor compared with intact wild-type roots. Data are means ± se (n > 12). Slow induction kinetics under actinic light are shown for Y_II (A–D), and other data (E–H) were obtained after actinic light exposure (110 µmol photons m⁻² s⁻¹) for 10 min. Samples at 21 d old were used for all experiments. Different letters indicate significant differences (P < 0.05, Tukey-Kramer multiple comparison test).

Figure 7.

Auxin regulates root greening independent of cytokinin signaling. A, Chl content in intact roots of the wild type and cytokinin signaling mutants in the absence (intact) or presence of 10 μm PCIB (intact, +PCIB). Different letters indicate significant differences (P < 0.05, Tukey-Kramer multiple comparison test). B, GFP fluorescence in roots of the TCSn::GFP line. For the top images, roots of 21-d-old intact seedlings were grown in the absence (untreated) or presence of 10 μm PCIB for 7 d. Aerial parts were detached just before observation. For the bottom images, 21-d-old root samples were detached from 14-d-old seedlings and grown on agar medium containing no or 1 μm indole-3-acetic acid (IAA). C, Gene expression analysis in 21-d-old detached root samples treated with or without 1 μm IAA for 7 d. Data are means ± se fold difference from the 21-d-old intact wild-type root samples after normalizing to ACTIN8 (n = 3). Asterisks indicate significant differences from the untreated control (*, P < 0.05 and **, P < 0.01, Student’s t test). D, Slow induction kinetics of Y_II in intact roots of 28-d-old seedlings grown in the absence or presence of 10 μm PCIB for 7 d (left graph) and root samples detached from 21-d-old seedlings and grown on agar medium containing no or 1 μm IAA for 7 d (right graph). Data are means ± se (n > 12). Actinic light intensity was 110 µmol photons m⁻² s⁻¹.

Figure 8.

Model of the regulatory pathways of root greening after shoot removal. Chloroplast development in roots is positively regulated by cytokinin signaling via the His kinases AHK2 and AHK3 and negatively regulated by auxin signaling via INDOLE-3-ACETIC ACID INDUCIBLE14 (IAA14) and AUXIN RESPONSE FACTORs (ARFs). In intact roots, auxin transported from the shoot represses chloroplast development, but in detached roots, the negative auxin regulation is attenuated, and the wound-responsive transcription factor WIND1 is induced in response to shoot removal and activates the type B ARR-mediated cytokinin signaling pathway. Several transcription factors associated with chloroplast development (HY5, GNC, GNL, and GLK2) function downstream of auxin/cytokinin signaling to directly and indirectly up-regulate nucleus- and plastid-encoded genes associated with chloroplast development. Type B ARRs also directly regulate some photosynthesis-associated nuclear genes and induce Chl accumulation and photosynthetic activation in roots. Arrows and bars represent positive and negative regulation, respectively.