Overexpression of copper/zinc superoxide dismutase from mangrove Kandelia candel in tobacco enhances salinity tolerance by the reduction of reactive oxygen species in chloroplast

Abstract

Na(+) uptake and transport in Kandelia candel and antioxidative defense were investigated under rising NaCl stress from 100 to 300 mM. Salinized K. candel roots had a net Na(+) efflux with a declined flux rate during an extended NaCl exposure. Na(+) buildup in leaves enhanced H2O2 levels, superoxide dismutase (SOD) activity, and increased transcription of CSD gene encoding a Cu/Zn SOD. Sequence and subcellular localization analyses have revealed that KcCSD is a typical Cu/Zn SOD in chloroplast. The transgenic tobacco experimental system was used as a functional genetics model to test the effect of KcCSD on salinity tolerance. KcCSD-transgenic lines were more Na(+) tolerant than wild-type (WT) tobacco in terms of lipid peroxidation, root growth, and survival rate. In the latter, 100 mM NaCl led to a remarkable reduction in chlorophyll content and a/b ratio, decreased maximal chlorophyll a fluorescence, and photochemical efficiency of photosystem II. NaCl stress in WT resulted from H2O2 burst in chloroplast. Na(+) injury to chloroplast was less pronounced in KcCSD-transgenic plants due to upregulated antioxidant defense. KcCSD-transgenic tobacco enhanced SOD activity by an increment in SOD isoenzymes under 100 mM NaCl stress from 24 h to 7 day. Catalase activity rose in KcCSD overexpressing tobacco plants. KcCSD-transgenic plants better scavenged NaCl-elicited reactive oxygen species (ROS) compared to WT ones. In conclusion, K. candel effectively excluded Na(+) in roots during a short exposure; and increased CSD expression to reduce ROS in chloroplast in a long-term and high saline environment.

Keywords: Kandelia candel; Na+ flux; catalase; hydrogen peroxide; salt; superoxide anion; superoxide dismutase.

Figure 1

Effects of NaCl on net Na⁺ fluxes in young roots of K. candel. Plants were treated by a rising NaCl concentration from 100 to 300 mM weekly for 3 weeks. The control was not exposed to NaCl. Steady Na⁺ flux profiles on week 1 (A), week 2 (B), and week 3 (C) were determined along root axis at apical zones, 200–2000 μm from the tip. Each point is the mean of 4–6 replicates. Bars represent standard error of the mean. ^*P < 0.05, control and NaCl treatment.

Figure 2

Effects of NaCl on Na⁺ concentrations in roots and shoots in K. candel. Plants were treated by a rising NaCl concentration from 100 to 300 mM weekly for 3 weeks. Each column is the mean of 4–6 replicates. Bars represent standard error of the mean. Different letters denote significant differences at P < 0.05 between treatments in each organ.

Figure 3

Total concentration of H₂O₂, total SOD activity, and KcCSD expression in K. candel leaves under rising NaCl stress. Plants were treated by a rising NaCl concentration from 100 to 300 mM weekly for 3 weeks. Each column is the mean of 4–6 replicates. Bars represent standard error of the mean. Different letters above columns represent significant differences at P < 0.05 during treatment.

Figure 4

Amino acid sequence and phylogenetic analysis of KcCSD. (A) Amino acid sequences of Cu/Zn SOD from Kandelia candle (KcCSD), Bruguiera gymnorrhiza (BgchlCSD), Dimocarpus longan (DlchlCSD), Gossypium hirsutum chloroplast (GhchlCSD), and Arabidopsis thaliana chloroplast (AtCSD2). Asterisks (^*) and dots (·, :) indicate identical and conserved amino acid residues, respectively. Italics are chloroplast transit peptide. Bolds indicate the conserved Cu²⁺ or Zn²⁺ binding site. Activity sites are underlined. (B) Neighbor-joined phylogenetic tree for CSD protein sequence (chloroplast CSDs with no chloroplast transit peptides) in various species. The alignment used for this analysis is available from the database (Supplementary Table S1). Different species acronyms are: Ah, Amaranthus hypochondriacus; Am, Avicennia marina; At, Arabidopsis thaliana; Bg, Bruguiera gymnorrhiza; Br, Brassica rapa subsp. Pekinensis; Cm, Chenopodiastrum murale; Dl, Dimocarpus longan; Ga, Gossypium arboreum; Gh, Gossypium hirsutum; Hb, Hevea brasiliensis; Hr, Haberlea rhodopenis; Hv, Hordeum vulgare; Kc, Kandelia candel; Pp, Prunus persica; Ps, Populus suaveolens; Pt, Populus tremuloides; Rs, Raphanus sativus; Vv, Vitis vinifera.

Figure 5

Chloroplast subcellular localization of KcCSD. Determined by transient expression of GFP alone (vector control; A–C) and a fusion KcCSD-GFP protein (KcCSD-GFP; D–F) in Arabidopsis protoplasts. Green fluorescence of GFP (A,D) and red auto-fluorescence of chlorophyll (B,E) were monitored separately using a confocal laser scanning microscope, and the two color fluorescence images (C,F) were merged. Bars = 10 μm.

Figure 6

NaCl tolerance of wild-type tobacco and KcCSD-transgenic lines. Seeds of wild-type (WT) and transgenic lines (L7 and L8) were germinated on MS medium for 7 days, then supplemented with 0 or 150 mM NaCl for another 7 days. (A,C,D) Show root growth and survival rate of tobacco seedlings. (B,E) Show plant performance and net photosynthetic rate of tobacco plants after exposure to 0 or 100 mM NaCl for 7 (E) or 14 (B) days. Prior to NaCl treatment, 4-weeks old rooted plants of WT and transgenic lines were transferred to 1/4 Hoagland's nutrient solution for 2-weeks acclimation. In (C–E), each column is the mean of 4–6 replicates. Bars represent standard error of the mean. Different letters above columns represent significant differences at P < 0.05 between WT and transgenic lines in control and NaCl treatments. Scale bars: A: 0.5 cm, B: 5 cm.

Figure 7

Effects of NaCl on chlorophyll content and fluorescence in wild-type tobacco and KcCSD-transgenic lines. Four-weeks old rooted plants of wild-type (WT) and transgenic lines (L7 and L8) were transferred to 1/4 Hoagland's nutrient solution for 2-weeks acclimation, then exposed to 0 or 100 mM NaCl for 7 days. (A) Chlorophyll content, (B) Chlorophyll a/b ratio, (C) Ratio of variable to maximal chlorophyll fluorescence (Fv/Fm), (D) Actual photochemical efficiency of PSII (Φ PSII). Each column is the mean of 4–6 replicates. Bars represent standard error of the mean. Different letters above columns represent significant differences at P < 0.05 between WT and transgenic lines in control and NaCl treatments.

Figure 8

Effects of NaCl on leaf ROS and malondialdehyde levels in wild-type tobacco and KcCSD-transgenic lines. Four-weeks old rooted plants of wild-type (WT) and transgenic lines (L7 and L8) were transferred to 1/4 Hoagland's nutrient solution for 2-weeks acclimation, then exposed to 0 or 100 mM NaCl for 7 days. Leaf discs (2 cm in diameter) were sampled. (A) In situ O⁻₂, (B) In situ H₂O₂, (C) Total H₂O₂, and (D) Malondialdehyde (MDA) content. In (C,D), each column is the mean of 4–6 replicates. Bars represent standard error of the mean. Different letters above columns represent significant differences at P < 0.05 between WT and transgenic lines in control and NaCl treatments.

Figure 9

Effect of NaCl on chloroplast H₂O₂ production in wild-type tobacco and KcCSD-transgenic lines. Seeds of wild-type (WT) and transgenic lines (L7 and L8) were germinated on MS medium for 7 days, then supplemented with 0 (A) or 150 mM NaCl (B) for 7 days. Seedlings treated with or without NaCl were stained with 10 μM H₂DCF-DA for H₂O₂ detection. Green fluorescence of H₂DCF-DA and red auto-fluorescence of chlorophyll were monitored separately using a confocal laser scanning microscope, and two color fluorescence images were merged. Bars = 20 μm.

Figure 10

Effect of NaCl on activities of antioxidant enzymes in wild-type tobacco and KcCSD-transgenic lines. Four-weeks old rooted plants of wild-type (WT) and transgenic lines (L7 and L8) were transferred to 1/4 Hoagland's nutrient solution for 2-weeks acclimation. Hydroponically acclimated plants were subjected to 0 or 100 mM NaCl for 24 h. (A) Total SOD activity, (B) CAT activity, and (C) APX activity. Each column is the mean of 4–6 replicates. Bars represent standard error of the mean. Different letters above columns represent significant differences at P < 0.05 between wild-type and transgenic lines in control and NaCl treatments.

Figure 11

Effect of NaCl on activities of SOD isoenzymes in leaves of wild-type tobacco and KcCSD-transgenic lines. Four-weeks old rooted plants of wild-type (WT) and transgenic lines (L7 and L8) were transferred to 1/4 Hoagland's nutrient solution for 2-weeks acclimation. Hydroponically acclimated plants were subjected to 0 or 100 mM NaCl for 7 days. The arrow indicates remarkably increased SOD isoform in transgenic lines.

Figure 12

Schematic model showing physiological mechanisms of K. candel adaptation to NaCl. Under a short-term NaCl treatment, K. candel roots extrude Na⁺ by the plasma membrane Na⁺/H⁺ antiport system driven by H⁺-ATPase. During a prolonged stress, K. candel roots were unable to effectively limit Na⁺ uptake and transport, leading to Na⁺ accumulation in shoots. The buildup of Na⁺ in leaves favors the formation of O⁻₂ via electron transport chain in chloroplasts. Salinized K. candel increases transcription of CSD gene encoding a copper/zinc superoxide dismutase (Cu/Zn SOD). An increased SOD activity in chloroplasts promotes the conversion of O⁻₂ to H₂O₂; subsequently, chloroplasts scavenge and eliminate H₂O₂ via the ascorbate-glutathione (ASC-GSH) cycle (Asada, 1999). Moreover, the activated antioxidant enzymes, such as catalase (CAT), and ascorbate peroxidase (APX), readily scavenge H₂O₂ when the molecule diffuses across thylakoid membranes to the cytosol. As a result, the efficient scavenging of Na⁺-elicited ROS in chloroplasts avoids an occurrence of oxidative burst, and thus alleviates chloroplast injury from excessive ROS under the stress.