Chloroplastic photoprotective strategies differ between bundle sheath and mesophyll cells in maize (Zea mays L.) Under drought

Abstract

Bundle sheath cells play a crucial role in photosynthesis in C4 plants, but the structure and function of photosystem II (PSII) in these cells is still controversial. Photoprotective roles of bundle sheath chloroplasts at the occurrence of environmental stresses have not been investigated so far. Non-photochemical quenching (NPQ) of chlorophyll a fluorescence is the photoprotective mechanism that responds to a changing energy balance in chloroplasts. In the present study, we found a much higher NPQ in bundle sheath chloroplasts than in mesophyll chloroplasts under a drought stress. This change was accompanied by a more rapid dephosphorylation of light-harvesting complex II (LHCII) subunits and a greater increase in PSII subunit S (PsbS) protein abundance than in mesophyll cell chloroplasts. Histochemical staining of reactive oxygen species (ROS) suggested that the high NPQ may be one of the main reasons for the lower accumulation of ROS in bundle sheath chloroplasts. This may maintain the stable functioning of bundle sheath cells under drought condition. These results indicate that the superior capacity for dissipation of excitation energy in bundle sheath chloroplasts may be an environmental adaptation unique to C4 plants.

Keywords: bundle sheath chloroplast; drought stress; maize (Zea mays L.); non-photochemical quenching; photoprotection; reactive oxygen species.

Figure 1

Measurement of reactive oxygen species (ROS) and lipid peroxidation in mesophyll (M) and bundle sheath (BS) cells of maize leaves under drought stress. Histochemical assays for O₂⁻ and H₂O₂ in leaves by nitro-blue tetrazolium (NBT; A) and 3,3-diaminobenzidine (DAB; B) staining, respectively. Distribution of O₂⁻ and H₂O₂ in M and BS cells were imaged in microscope. The microscopic images in M and BS cells were quantitative analyzed (C,D). Plastid malondialdehyde (MDA) content (E) in M and BS chloroplasts of maize leaves were detected. CK, S1, S2, and S3 represent, respectively, the soil moisture regimes of well-watered, mild drought stress, moderate drought stress and severe drought stress. Values are expressed as the means ± SD from four independent biological replicates (n = 4). Different letters depict significant differences between the treatments (p < 0.05) according to Duncan’s multiplication range test.

Figure 2

Chlorophyll fluorescence imaging and light response curves (LRCs) in intact maize leaves under drought stress. Fv/Fm, the maximal photochemical quantum yield of PSII in darkness; Fv’/Fm’, the maximal photochemical quantum yield of PSII under light; Φ_PSII, the effective photochemical quantum yield of PSII under light; NPQ, the non-photochemical fluorescence quenching under light. CK, well-watered control; S1, mild drought stress; S2, moderate drought stress; and S3, severe drought stress. Values beside the individual images present quantitative means ± SD (n = 4). Vertical bars represent SD of the mean (n = 4).

Figure 3

Chlorophyll fluorescence imaging and LRCs in M and BS chloroplasts of maize leaves under drought stress. Fv/Fm, the maximal photochemical quantum yield of PSII in darkness; Fv’/Fm’, the maximal photochemical quantum yield of PSII under light; Φ_PSII, the effective photochemical quantum yield of PSII under light; NPQ, the non-photochemical fluorescence quenching under light. CK, well-watered control; S1, mild drought stress; S2, moderate drought stress; and S3, severe drought stress. Vertical bars represent SD of the mean (n = 4). Different letters mean significant differences at the 0.05 level according to Duncan’s multiplication range test.

Figure 4

Assays of non-photochemical quenching (NPQ) kinetics under drought stress. (A) Measurement of NPQ kinetics in intact maize leaves. (B) Measurement of NPQ kinetics in M and BS chloroplasts of maize leaves. The two consecutive periods of illumination with 1,500 mol m⁻² s⁻¹ for 500 s with a 500 s period of darkness in between, as indicated by the white (light on) and black (dark) bars at the bottom of figure. CK, well-watered control; S1, mild drought stress; S2, moderate drought stress; and S3, severe drought stress. Vertical bars represent SD of the mean (n = 4).

Figure 5

Reversible phosphorylation of PSII proteins in M and BS chloroplasts of maize leaves under drought stress. (A) Immunoblot analysis of the PSII proteins phosphorylation in M and BS chloroplasts under drought stress. Proteins in M (1.0 μg Chl) and BS chloroplasts (1.5 μg Chl), were detected with anti-PThr antibody. (B) Coomassie staining of protein samples (CBS) was shown as a control. The positions of detected phosphoproteins and molecular masses of protein markers (in kDa) are indicated. (C–E) Dephosphorylation of PSII proteins in vivo under severe drought stress. Maize leaves were illuminated 120 min at 25°C and then transferred to darkness and incubated at 25°C. Dephosphorylation was terminated at the indicated time points by freezing the leaves in liquid nitrogen. Thylakoid membranes in M and BS cells were isolated and the extent of protein phosphorylation was determined using anti-PThr antibody. Before conducting the dephosphorylation experiments, different light intensities were used for induction of higher in vivo phosphorylation levels of either core proteins or LHCII. Maize leaves were illuminated under a PFD 1,000 μmol photons m⁻² s⁻¹ for more effective phosphorylation of PSII core proteins (C,D) or under a PFD 80 μmol photons m⁻² s⁻¹ for induction of LHCII phosphorylation (E). CK, well-watered control; S1, mild drought stress; S2, moderate drought stress; and S3, severe drought stress. The results shown are representative of those obtained in at least three independent experiments. Thylakoids were isolated in the presence of 10 mM NaF.

Figure 6

Steady-state levels of PSII proteins in M and BS thylakoids of maize leaves under drought stress. Immunoblot analyses of PSII proteins in M thylakoids (1.0 μg Chl) and BS thylakoids (1.5 μg Chl) were performed using antibodies specific for D1, D2, CP43, CP47, Lhcb1, Lhcb2, and PsbS. The results shown are representative of those obtained in at least three independent experiments. CK, S1, S2, and S3 represent, respectively, the well-watered, mild drought, moderate drought and severe drought treatments.

Figure 7

The composition of protein complexes in M and BS thylakoids of maize leaves under drought stress. (A) Membranes (15 μg Chl) in M thylakoids were solubilized with 1% n-dodecyl β-D-maltoside (DDM) and loaded onto 4%–12% acrylamide Blue-Native gel. (B) Membranes (15 μg Chl) in BS thylakoids were solubilized with 2% DDM and loaded onto 4%–12% acrylamide Blue-Native gel. The bands of BN-PAGE were confirmed by immunoblotting with CP43, D1, Lhcb1, and PsaA specific antibodies (on the right). The control line of the BN gel was selected for immunodetection. The results shown are representative of those obtained in at least three independent experiments. CK, S1, S2, and S3 represent, respectively, the well-watered, mild drought, moderate drought and severe drought treatments.

Figure 8

Transmission electron microscope analysis of chloroplasts in maize leaves exposed to progressive drought stress. GL and SL represent, respectively, the grana lamellae and stroma lamellae of mesophyll thylakoids, and SG represents starch grain. CK, well-watered control; S1, mild drought stress; S2, moderate drought stress; and S3, severe drought stress.