Melatonin-Regulated Chaperone Binding Protein Plays a Key Role in Cadmium Stress Tolerance in Rice, Revealed by the Functional Characterization of a Novel Serotonin N-Acetyltransferase 3 (SNAT3) in Rice

Abstract

The study of the mechanisms by which melatonin protects against cadmium (Cd) toxicity in plants is still in its infancy, particularly at the molecular level. In this study, the gene encoding a novel serotonin N-acetyltransferase 3 (SNAT3) in rice, a pivotal enzyme in the melatonin biosynthetic pathway, was cloned. Rice (Oryza sativa) OsSNAT3 is the first identified plant ortholog of archaeon Thermoplasma volcanium SNAT. The purified recombinant OsSNAT3 catalyzed the conversion of serotonin and 5-methoxytryptamine to N-acetylserotonin and melatonin, respectively. The suppression of OsSNAT3 by RNAi led to a decline in endogenous melatonin levels followed by a reduction in Cd tolerance in transgenic RNAi rice lines. In addition, the expression levels of genes encoding the endoplasmic reticulum (ER) chaperones BiP3, BiP4, and BiP5 were much lower in RNAi lines than in the wild type. In transgenic rice plants overexpressing OsSNAT3 (SNAT3-OE), however, melatonin levels were higher than in wild-type plants. SNAT3-OE plants also tolerated Cd stress, as indicated by seedling growth, malondialdehyde, and chlorophyll levels. BiP4 expression was much higher in the SNAT3-OE lines than in the wild type. These results indicate that melatonin engineering could help crops withstand Cd stress, resulting in high yields in Cd-contaminated fields.

Keywords: binding proteins; cadmium tolerance; chaperone; melatonin; serotonin N-acetyltransferase; transgenic rice.

Figure 1

(A) Amino acid sequence alignment between archaeon TvSNAT and rice OsSNAT3. The conserved acetyl coenzyme A binding sites are underlined and key residues for SNAT activity are shown in bold. Stars indicate identical amino acids; dashes denote gaps. (B) Phylogenetic tree of OsSNAT3 among multiple SNAT genes in rice. The scale bar represents 0.8 substitutions per site. GenBank accession numbers are NC_003413 (TvSNAT), AK059369 (OsSNAT1), AK068156 (OsSNAT2), and AK241100 (OsSNAT3). (C) Purification of His6-tagged OsSNAT3 proteins. E. coli BL21 (DE3) cells harboring pET300-OsSNAT3 and pET28b-OsSNAT3 plasmids were induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) for 5 h at 28 °C. M, molecular mass standards. Lane 1, total proteins in 15 µL bacterial culture without IPTG; lane 2, total proteins in 15 µL bacterial culture with IPTG; lane 3, 30 µg soluble protein; lane 4, 5 µg affinity-chromatography-purified protein. (D) SNAT activity measured in purified N-terminal His6-tagged OsSNAT3 and purified C-terminal His6-tagged OsSNAT3. Protein samples were separated by SDS-PAGE on a 12% polyacrylamide gel and stained with Coomassie blue.

Figure 2

Enzyme kinetics of OsSNAT3. SNAT activity as a function of (A) pH and (B) temperature, and determination of K_m and V_max values of OsSNAT3 using (C) serotonin and (D) 5-methoxytryptamine (5-MT) as substrates. Recombinant purified OsSNAT3 (1 µg) was assayed in the presence of different serotonin and 5-MT concentrations for 1 h at different temperatures and pH values, followed by high-performance liquid chromatography detection of N-acetylserotonin (NAS) and melatonin. Kinetic values of K_m and V_max were determined using Lineweaver–Burk plots. Values are presented as the mean ± SD (n = 3). nd, not detected.

Figure 3

SNAT reactions and substrate preferences. (A) SNAT activity toward serotonin and 5-MT substrates. (B) SNAT activity toward other substrates. (C) Activity of recombinant purified OsSNAT3 toward serotonin (0.5 mM) and various amines (0.5 mM) at 55 °C and pH 8.8. Values are presented as the mean ± SD (n = 3). Different letters indicate significant differences vs. the wild type (Tukey’s HSD; p < 0.05).

Figure 4

Subcellular localization of OsSNAT3. (A) Red fluorescence of OsSNAT3-mCherry. (B) Green fluorescence of cytoplasmic green fluorescent protein (GFP). (C) Merged fluorescence images (A + B). Thirty-day-old tobacco seedlings infiltrated with Agrobacterium tumefaciens (GV2260) containing XVE-inducible OsSNAT3-mCherry or constitutive 35S:GFP (cytosolic marker). Bars = 10 μm.

Figure 5

Generation of OsSNAT3 RNAi transgenic rice and the melatonin content of rice seedlings. (A) RNAi binary vector used for OsSNAT3 suppression. (B) Phenotypes of 7-day-old rice seedlings. (C) RT-PCR analyses of transgenic and wild-type 7-day-old rice seedlings. (D) Photograph of 7-day-old rice seedlings treated for 3 days with 0.5 mM CdCl₂. (E) Melatonin contents of 7-day-old rice seedlings treated for 3 days with 0.5 mM CdCl₂. Ubi-P, maize ubiquitin promoter; HPT, hygromycin phosphotransferase; WT, wild type; UBQ5, rice ubiquitin 5 gene. GenBank accession numbers of SNAT1, SNAT2, SNAT3, and UBQ5 are AK059369, AK068156, AK241100, and AK061988, respectively. Different letters indicate significant differences vs. the wild type (Tukey’s HSD; p < 0.05).

Figure 6

Enhanced Cd stress susceptibility in OsSNAT3 RNAi transgenic rice plants. (A) Growth phenotype, (B) shoot length, (C) root length, (D) malondialdehyde (MDA) contents, and (E) melatonin content in 7-day-old rice seedlings. (F) Gene expression profiles determined by RT-PCR in Cd-stressed rice plants. Dehusked seeds were surface-sterilized and transferred for 7 days to half-strength Murashige Skoog (MS) medium containing 0.5 mM CdCl₂ under a 14 h light/10 h dark photoperiod and an incubation temperature of 28 °C/24 °C (day/night). Values are presented as the mean ± SD (n = 3). Different letters indicate significant differences vs. the wild type (Tukey’s HSD; p < 0.05). GenBank accession numbers: PDIL1–1 (AK068268), SODA1 (AAA62657), APX1 (AB050724), APX4 (AK104490), CatB (AK069446), CNX (AK069118), BiP1 (AK119653), BiP2 (BAS86012), BiP3 (BAS93656), BiP4 (AK106696), BiP5 (BAF23108), SGT1 (BAF05534), GR2 (BAF10399), and UBQ5 (AK061988).

Figure 7

Enhanced Cd tolerance in OsSNAT3-overexpressing transgenic rice plants. (A) Schematic diagram of the binary vector for OsSNAT3 overexpression. (B) Phenotype of Cd-treated 7-day-old rice seedlings. (C) Shoot length, (D) root length, and (E) chlorophyll, (F) MDA, and (G) melatonin contents in Cd-treated plants. (H) Gene expression profiles of Cd-treated rice plants, as determined by RT-PCR. (I) BiP4 expression levels determined in a quantitative real-time PCR analysis of Cd-stressed rice plants. OsSNAT3, Oryza sativa serotonin N-acetyltrasferase3; Ubi-P, maize ubiquitin promoter; HPT, hygromycin phosphotransferase; WT, wild type; UBQ5, rice ubiquitin 5 gene. GenBank accession numbers are listed in Figure 6. Different letters indicate significant differences vs. the wild type (Tukey’s HSD; p < 0.05).

Figure 8

Phylogenetic tree of rice SNAT3 (OsSNAT3) shown as red words, an archaeal SNAT ortholog. The scale bar represents 0.3 substitutions per site. GenBank accession numbers are as follows: Arabidopsis Naa50 (NM_121172), rice SNAT3 (AK241100), human Naa50 (BAB14397), Chlamydomonas arylalkylamine N-acetyltransferase (AANAT) (AB474787), human AANAT (NP_001079), sheep AANAT (NP_001009461), archaea SNAT (NC_002689), pine SNAT1 (PSY00020345), E. coli RimI (WP_137442509), Arabidopsis SNAT1 (At1g32070), rice SNAT1 (AK059369), rice SNAT2 (AK068156), porphyra SNAT (NC_007932), cyanobacteria SNAT (NP_442603), grapevine SNAT2 (RVX06207), and Arabidopsis SNAT2 (At1g26220).