The Expression of Manganese Superoxide Dismutase Gene from Nelumbo nucifera Responds Strongly to Chilling and Oxidative Stresses

Structure of the sacred lotus NnMSD1 gene

The full length cDNA of NnMSD1 contains an open reading frame (ORF) of 699 nucleotides, flanked by 35 bp of a non-coding sequence at the 5′ end and 251 bp of a non-coding sequence with a 21 bp poly (A) tail at the 3′ end (Figure 1). Kozak consensus sequence was identified in the 5′-non-coding sequence, which may facilitate the initial binding of mRNA to the small submit of the ribosome. A comparison with the sequence of the NnMSD1 gene and its cDNA sequence indicated that six exons and five introns were present in the gene. The introns have a high AT content (>60%). The Intron 2 and Intron 3 are 102 bp and 142 bp, respectively, which are much shorter than the other three Introns (Table 1). The cDNA of NnMSD1 encodes a protein of 233 amino acid residues with a predicted molecular mass of 25.9 kDa and an isoelectric point of 6.6.

Comparison of the amino acid sequences of Mn-SOD genes from different species and analysis of conserved residues

Alignment of the deduced amino acid sequence of Mn-SOD from sacred lotus (AAX22235) with those from the other plants indicates that the N-terminal regions are less conserved (Figure 2). The sequences of Mn-SOD from Triticum aestivum (AAX68501), Pisum sativum (CAA42737), Zea mays (AAA72020), Oryza sativa (Q43008), Camellia sinensis (AAT68778), Prunus persica (CAB56851), Arabidopsis thaliana (AAC24832), Nicotiana plumbaginifolia (CAA32643) and sacred lotus share similarities that range from 73% to 78%. In addition, the sequences of Fe-SOD from P. sativum (CAD42655), Z. mays (BAD89495), A. thaliana (NP849441), O. sativa (XP550626), Lycopersicon esculentum (AAQ18699), Glycine max (AAQ13492), Vigna unguiculata (AAF28773) and Mn-SOD from sacred lotus share similarities that range from 25% to 30%.

Although plant Mn-SOD N-terminal regions are poorly conserved, they have the general features of mitochondrial targeting sequences, which direct Mn-SOD proteins to the mitochondrion. The N-terminal sequence of NnMSD1 shares common characteristics with mitochondrial transit peptides, such as an abundance of basic residues and the complete absence of acidic residues. Based on signal PV2.0 (Nielsen et al. 1997) and TARGET PV1.0 (Emanuelsson et al. 2000) prediction to N-terminal peptides, the deduced sacred lotus peptide has an N-terminal signal peptide of 24 amino acids (MALRSVISRKTLGSLGLGFSHARG) for mitochondrial subcellular localization. The Mn-SOD sequences contain four perfectly conserved regions in all plant species, namely consensus I to IV (Figure 1). Homology is most conserved in the domains corresponding to α-helices one (consensus V) and two (consensus III). In addition, the amino acids involved in the coordination of the metal ions are highly conserved (Borgstahl et al. 1992). After removing the 24 amino acid signal peptides, the predicted mature sacred lotus Mn-SOD and the other plant Mn-SODs share sequence similarities ranging from 77% to 81%. The predicted mature NnMSD1 showed highest homology (81%) to the Mn-SODs from C. sinensis, P. persica, and N. plumbaginifolia. The mature sacred lotus Mn-SOD protein has a calculated molecular mass of approximately 23.4 kDa and a pI of 5.9.

Phylogenetic analysis of Mn-SODs from different plant species revealed two subgroups (Figure 2). It should be noted that all Mn-SODs from cereals are grouped in the same subgroup. Subgroup 1 consists of the sequences from P. sativum, C. sinensis, A. thaliana, P. persica, N. plumbaginifolia, N. nucifera, Avicennia marina, Capsicum annuum, Euphorbia esula, and Zantedeschia aethiopica.

Genomic organization of NnMSD1 gene

In order to determine the copy number of the NnMSD1 gene, the genomic DNA (5 μg) of sacred lotus leaves was digested with EcoRI, HindIII, or BglII and hybridized with a 423 bp ³²P-labeled probe. Two strongly hybridizing bands were detected for each restriction enzyme (Figure 3). These data indicate that there are two NnMSD1 genes in the sacred lotus genome.

Expression of NnMSD1 gene in developing and germinating seeds

The development pattern of sacred lotus seeds is similar to that of most dicot seeds. There are three stages in the sacred lotus seed development: tissue differentiation (0–13 d after pollination, DAP), cell expansion (14–20 DAP) and desiccation-maturation stage (21–30 DAP). The expression patterns of the NnMSD1 gene were investigated during seed development by reverse transcriptase-polymerase chain reaction (RT-PCR) (Figure 4A). Strong and constant levels of NnMSD1 expression were detected in sacred lotus embryonic axes during seed development. The cotyledons showed low NnMSD1 gene expression on the 15 DAP, whereas there was no detectable NnMSD1 transcript in the cotyledons of 20 DAP and 25 DAP (Figure 4A).

The expression profile of the NnMSD1 gene during sacred lotus seed germination was examined using gene-specific primers (Figure 4B). NnMSD1 transcript was detected in mature dry embryonic axes. Within 24 h after imbibition, the transcript levels of the NnMSD1 gene in sacred lotus embryonic axes were the same as in the dry embryonic axes. However, its expression decreased obviously in the 30 h and 48 h after imbibition, and was restored in the 72 h after imbibition (Figure 4B).

Expression patterns of the NnMSD1 gene in germinated embryonic axes under various stress conditions

The expression patterns of the NnMSD1 gene in germinated sacred lotus embryonic axes under various stresses, such as heat shock (42 °C), chilling (4 °C), and exposure to stress-related chemicals, were investigated by RT-PCR. The stress-related chemicals include polyethylene glycol (PEG)-6000 (20% w/v), H₂O₂ (1.5%), NaCl (300 mmol/L), and abscisic acid (ABA) (100 μmol/L). The results of RT-PCR analysis show that NnMSD1 in the germinated embryonic axes responded strongly to chilling and oxidative stresses. However, NnMSD1 gene expression in germinated embryonic axes was inhibited by heat-shock and NaCl. No obvious effects of ABA and PEG on the NnMSD1 gene expression were observed (data not shown).

To get more information about the expression of NnMSD1 under chilling and oxidative stresses, real-time PCR was used to study the transcription of NnMSD1 after chilling and 1.5% of H₂O₂ treatments, respectively. For studying the effects of chilling on the NnMSD1 mRNA levels, 3-d-old germinated sacred lotus seeds were treated with chilling (4 °C) for 2, 6, 12, 24 and 48 h, respectively. NnMSD1 was strongly expressed after 2–48 h chilling treatment (Figure 5A).

To study NnMSD1 gene response to H₂O₂, the 3-d-old germinated seeds were incubated separately in aqueous solutions containing 1.5% of H₂O₂ for 2, 6, 12, 24 and 48 h, respectively. The results suggested that the expression of NnMSD1 in the germinated embryonic axes was inhibited after 2, 6 and 48 h treatment, but stimulated significantly after 12 and 24 h treatment (Figure 5B).

Plant materials and treatments with exogenous factors

Seeds of sacred lotus (Nelumbo nucifera cv. Hongtoujin) were collected from plants grown in ponds in Sanshui Lotus World, Guangdong Province, China. Developing embryonic axes were used for gene cloning and RNA preparation in the study of gene expression during seed development. Mature seeds were used in germination test. Seeds were germinated in distilled water at 25 °C for 0, 1, 2, and 3 d. Seeds that germinated for 3 d were subjected to various stress treatments by submerging in aqueous solutions containing PEG 6000 (20% w/v), H₂O₂ (1.5%), NaCl (300 mmol/L), and ABA (100 μmol/L) for 24 h or incubating at different temperatures (heat shock at 42 °C and low temperature at 4 °C) for up to 48 h. All treated materials were immediately frozen in liquid nitrogen and stored at −70 °C until use.

RNA preparation and RACE

Total RNA was isolated from developing embryonic axes by using Trizol Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Adapter ligated cDNA was synthesized using the SMART rapid amplification of cDNA ends (RACE) cDNA amplification kit (BD Biosciences, San Jose, CA, USA) according to the product manual. 5′-RACE was carried out using 5′ primer and the degenerate primer (DP: 5′-AGGTAGTAA/CGCATGCTCCCA-3′) designed from the conserved region of the plant SODs. 3′-RACE was carried out using CDSIII /3′ PCR primer and the gene specific primer (GSP1: 5′-TTGAAGCATTGGTGAAAAAGG-3′) designed according to sequence of the 680 bp fragment. PCR products were purified and sequenced. As the first strand cDNA was synthesized with the SMART cDNA synthesis kit, only one gene-specific primer was used for 5′ RACE based on the conserved domain WEHAYY of Mn-SOD. The 5′ end RACE product was recovered and sequenced directly. A gene-specific primer was designed for 3′ RACE according to the acquired 5′ fragment of the NnMSD1 gene from sacred lotus cDNA. After the two sequences were assembled, the full length cDNA of NnMSD1 was obtained.

DNA and PCR amplification

Genomic DNA was extracted from sacred lotus leaves according to the protocol described by Pich and Schubert (1993). Two gene-specific primers GSP2 (5′-GGCGCTGATCAAATTGGAAG-3′) and GSP3 (5′-GAAGACTGCCTGCCCAACTC-3′) were designed based on the sequence of the acquired NnMSD1 cDNA to amplify the corresponding genomic DNA sequence. The PCR product of 3469 bp was sequenced directly.

Analysis of the DNA sequence

The homologous sequences of the gene NnMSD1 were searched using BLAST (Altschul et al. 1990) and the sequence alignments were carried out using the Cluster W program (Thompson et al. 1994). The intron position of NnMSD1 gene was determined by comparison of the genomic DNA sequence with the corresponding cDNA sequence.

Southern blot analysis

Five micrograms of genomic DNA was digested with the restriction endonucleases EcoRI, HindIII, and BglII at 37 °C overnight, and the resulting fragments were resolved in a 0.8% agarose gel and transferred onto a piece of nylon membrane (Hybond-N⁺, Amersham Biosciences, Uppsala, Sweden). The blotted membrane was UV cross-linked and hybridized with a [³²P]-labeled probe from NnMSD1 (position 1 281–1 703). In the high stringency condition, the membrane was hybridized at 42 °C for 14 h and washed twice with 0.1% sodium dodecyl sulfate (SDS) in 2 × standard saline citrate (SSC) (150 mmol/L NaCl, 15 mmol/L sodium citrate) solution for 15 min, and then twice with 0.1% SDS in 0.2 × SSC solution for 15 min at 42 °C. Signals were quantified using a Molecular Imager FX (Bio-Rad, Hercules, CA, USA).

RT-PCR and real-time PCR analysis

Total RNA was exacted from the embryonic axes and cotyledons by using Trizol Reagent (Invitrogen), according to the manufacturer's instructions. First strand cDNA was synthesized with total RNA using the TaKaRa (Shiga, Japan) RNA PCR Kit (AMV) Ver. 3.0. The relative levels of Mn-SOD were measured by RT-PCR using N. nucifera 18s rRNA as an internal standard. Second-strand cDNA synthesized and PCR amplification with Mn SOD gene-specific primers (forward primer: 5′-CCATCACCAGAAGCACCATC-3′; reverse primer: 5′-TTTAACCACGCCTAGCCACA-3′) were carried out using the following conditions: 28 cycles of denaturaton at 94 °C for 30 s, annealing at 58 °C for 1 min and extension at 68 °C for 1.5 min. The amplification of the internal standard 18s rRNA cDNA was carried out in a similar manner, but with 22 cycles using two 18s rRNA-specific primers (forward primer: 5′-CCATAAACGATGCCGAC-3′, reverse primer: 5′-CACCACCCATAGAATCAAGA-3′). RT-PCR product was separated on a 2% agarose gel and visualized by ethidium bromide staining. The expected size of RT-PCR product of the NnMSD1 gene was 325 bp. Real-time PCR was carried out and analyzed with an IQ5 Multicolor Real time PCR Detection System (Bio-Rad) following the manufacturer's instructions with another NnMSD1 gene-specific primer (forward primer: 5′-TTATGCGGCTCCATCACCA-3′; reverse primer: 5′-TAACCACTGCGGACGAATC-3′). The program was 95 °C denaturation for 30 s, 40 cycles of 95 °C for 10 s, 56 °C for 15 s, and 72 °C for 20 s.

(Handling editor: Lizhe An)