Photosynthesis of root chloroplasts developed in Arabidopsis lines overexpressing GOLDEN2-LIKE transcription factors

Abstract

In plants, genes involved in photosynthesis are encoded separately in nuclei and plastids, and tight cooperation between these two genomes is therefore required for the development of functional chloroplasts. Golden2-like (GLK) transcription factors are involved in chloroplast development, directly targeting photosynthesis-associated nuclear genes for up-regulation. Although overexpression of GLKs leads to chloroplast development in non-photosynthetic organs, the mechanisms of coordination between the nuclear gene expression influenced by GLKs and the photosynthetic processes inside chloroplasts are largely unknown. To elucidate the impact of GLK-induced expression of photosynthesis-associated nuclear genes on the construction of photosynthetic systems, chloroplast morphology and photosynthetic characteristics in greenish roots of Arabidopsis thaliana lines overexpressing GLKs were compared with those in wild-type roots and leaves. Overexpression of GLKs caused up-regulation of not only their direct targets but also non-target nuclear and plastid genes, leading to global induction of chloroplast biogenesis in the root. Large antennae relative to reaction centers were observed in wild-type roots and were further enhanced by GLK overexpression due to the increased expression of target genes associated with peripheral light-harvesting antennae. Photochemical efficiency was lower in the root chloroplasts than in leaf chloroplasts, suggesting that the imbalance in the photosynthetic machinery decreases the efficiency of light utilization in root chloroplasts. Despite the low photochemical efficiency, root photosynthesis contributed to carbon assimilation in Arabidopsis. Moreover, GLK overexpression increased CO₂ fixation and promoted phototrophic performance of the root, showing the potential of root photosynthesis to improve effective carbon utilization in plants.

Keywords: Arabidopsis root; Chloroplast development; Construction of photosynthetic systems; GLK; Photosynthesis.

Fig. 1

Chl accumulation in GLK_OX roots. (A) Color phenotype of roots of 21-day-old plants. GLK_OX plants have greenish roots compared with the yellowish wild-type and albino hy5-215 roots. (B) Confocal microscopy of Chl fluorescence in the primary root. Chl autofluorescence micrographs were merged with differential interference contrast images. Arrows indicate boundaries between the stele and endodermis. Bar = 50 µm.

Fig. 2

Plastid development in GLK_OX roots. (A–C) Plastid ultrastructure in the primary root cells of wild-type (WT) (A), GLK1_OX (B) and GLK2_OX seedlings (C). (D) A typical chloroplast in a mature Arabidopsis leaf cell. Bars = 0.5 µm. (E) Histogram of plastid size in the outer cells of the stele determined from primary root cross-sections from WT and GLK_OX seedlings (n = 42, 87 and 64 for the WT, GLK1_OX, and GLK2_OX, respectively). (F) Histogram of plastid number in the outer cells of the stele determined from primary root cross-sections (n = 60, 45, and 54 for the WT, GLK1_OX, and GLK2_OX, respectively).

Fig. 3

Quantitative RT–PCR analysis of chloroplast biogenesis-associated gene expression in roots of wild-type and GLK_OX seedlings. Expression levels of nuclear-encoded genes for Chl biosynthesis and light harvesting (A), plastid-encoded genes (B), nuclear-encoded genes for sigma factors (C) and GATA-type nuclear transcription factor genes (D). Data are presented as the fold difference from wild-type root samples after normalization to the reference gene ACTIN8. Values are the means ± SE from three independent experiments.

Fig. 4

Differential accumulation of membrane photosynthetic proteins in roots of GLK1_OX and GLK2_OX. Immunoblot analysis of photosynthetic proteins in 10 µg of total membrane protein from root samples of wild type (WT), GLK1_OX (1_OX) and GLK2_OX (2_OX) compared with those in a dilution series (5, 1 and 0.2 µg) of total membrane protein from WT leaves.

Fig. 5

Comparison of PS complexes in roots of the wild type and GLK_OX lines with those in wild-type leaves. Characteristics of PS complexes were examined in samples from roots of wild type (WT R), GLK1_OX (GLK1_OX R) and GLK2_OX (GLK2_OX R) and from wild-type leaves (WT L). (A) Chl fluorescence emission spectra at 77K. Vertical dashed lines represent emission peaks at 684, 691 and 732 nm observed in WT L samples. Slight blue shifts in these peaks were observed in all root samples. Representative data from multiple independent experiments are shown (n > 3). (B and C) Transient fluorescence induction kinetics of Chl in the absence (B) or presence (C) of 40 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Values are means from three independent experiments. Two inflections, labeled J and I, are observed between the levels O (origin) and P (peak) only in the absence of DCMU (B).

Fig. 6

Maximum quantum yield of PSII (F_v/F_m). F_v/F_m levels were compared between leaves and roots from the wild type, GLK1_OX and GLK2_OX. Values are means ± SE (n = 5 for leaves and 7 for roots in each line).

Fig. 7

Light–response curves of Chl fluorescence parameters. Wild-type leaves (WT L) and roots of the wild type (WT R), GLK1_OX (GLK1_OX R) and GLK2_OX (GLK2_OX R) were dark adapted for 5 min prior to the measurements and exposed for 3 min to each light intensity. (A) Effective quantum yield of PSII (Φ_II). (B) Coefficient of photochemical quenching (qP), a measure of the redox state of the PSII acceptor side. (C) Maximum PSII quantum yield under light conditions (F_v′/F_m′). (D) Quantum yield of non-regulated energy dissipation (Φ_NO). (E) Quantum yield of regulated energy dissipation (Φ_NPQ). (F) Coefficient of non-photochemical quenching (qN). Data are means of multiple experiments (n = 5 for wild-type leaves and 7 for each root sample).

Fig. 8

Contribution of photoinhibition to total non-photochemical quenching (qN). Wild-type leaves (WT L) and roots of the wild type (WT R), GLK1_OX (GLK1_OX R) and GLK2_OX (GLK2_OX R) were dark adapted for 15 min and then exposed to light stress (420 µmol photons m⁻² s⁻¹) for 10 min. First, total qN was measured at the end of the light stress. After relaxation of the rapidly reversible qN component with additional dark treatment for 15 min, the remaining qN component was determined as the photoinhibition-related qN component (qI). Values are the means ± SE from three independent experiments.

Fig. 9

Quantum yield of PSI. Slow induction kinetics of the PSI quantum yields in wild-type leaves (WT L), and GLK1_OX leaves (GLK1_OX L) and roots (GLK1_OX R) were measured for 10 min under actinic light (126 µmol photons m⁻² s⁻¹). (A) Photochemical quantum yield of PSI (Φ_I). (B and C) Non-photochemical quantum yield of energy dissipation due to PSI donor-side limitation (Φ_ND) (B) and PSI acceptor-side limitation (Φ_NA) (C). Data are means of two independent measurements.

Fig. 10

Photosynthetic activity of roots. (A) Light–response curve of net O₂ evolution activity in roots. The zero level represents the light compensation point. (B) Net CO₂ fixation activity in roots under dark or light (200 µmol photons m⁻² s⁻¹) conditions. Numbers in the graph indicate the gross CO₂ fixation rate (µmol CO2 min⁻¹ g FW⁻¹) in each root sample calculated by subtracting the CO₂ fixation rate in the dark from that in the light. (C) Change in dry weight of detached roots. Roots detached from 21-day-old seedlings were further incubated for 7 d without a carbon source under dark or light (60 µmol photons m⁻² s⁻¹) conditions. The dry weight of the 28-day-old detached roots was normalized to the initial weight of the 21-day-old root. Values are means ± SE from three independent experiments for A and B, and from seven independent experiments for C.