The antioxidative defense system is involved in the premature senescence in transgenic tobacco (Nicotiana tabacum NC89)

Abstract

Background: α-Farnesene is a volatile sesquiterpene synthesized by the plant mevalonate (MVA) pathway through the action of α-farnesene synthase. The α-farnesene synthase 1 (MdAFS1) gene was isolated from apple peel (var. white winter pearmain), and transformed into tobacco (Nicotiana tabacum NC89). The transgenic plants had faster stem elongation during vegetative growth and earlier flowering than wild type (WT). Our studies focused on the transgenic tobacco phenotype.

Results: The levels of chlorophyll and soluble protein decreased and a lower seed biomass and reduced net photosynthetic rate (Pn) in transgenic plants. Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide radicals (O 2 (·-) ) had higher levels in transgenics compared to controls. Transgenic plants also had enhanced sensitivity to oxidative stress. The transcriptome of 8-week-old plants was studied to detect molecular changes. Differentially expressed unigene analysis showed that ubiquitin-mediated proteolysis, cell growth, and death unigenes were upregulated. Unigenes related to photosynthesis, antioxidant activity, and nitrogen metabolism were downregulated. Combined with the expression analysis of senescence marker genes, these results indicate that senescence started in the leaves of the transgenic plants at the vegetative growth stage.

Conclusions: The antioxidative defense system was compromised and the accumulation of reactive oxygen species (ROS) played an important role in the premature aging of transgenic plants.

Keywords: Reactive oxygen species (ROS); Senescence; Tobacco; Transcriptome.

Fig. 1

Identification of transgenic plants by qRT-PCR and GC–MS. a GC–MS analysis of terpenes of WT plants. b GC–MS analysis of terpenes of transgenic plants. c MdAFS1 transcript in different transgenic plants. d MdAFS1 transcript in different parts of transgenic plants. The MdAFS1 transcript level was normalized to 18S RNA expression. The standard error of the mean of three biological replicates (nested within three technical replicates)

Fig. 2

Phenotypic variation of wild-type (WT) and transgenic plants at different developmental stages. A Variation in the height of WT and transgenic plants at different developmental stages, each line is the mean of five replicates. Different letters indicate statistically significant differences at P ≤ 0.05, B eight-leaf period, and C filling period

Fig. 3

Measurement of senescence-related physiological parameters in WT and transgenic plants. A Chlorophyll content, B carotenoid content, C soluble protein content, D net photosynthetic rate, E shows water content, and F signifies malondialdehyde (MDA) content. Data are mean ± SE (n = 5, five biological replicates per line). Different letters indicate statistically significant differences at P ≤ 0.05

Fig. 4

Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of photosynthesis. Green represents downregulated unigenes (P ≤ 0.01, ratio ≤0.5). psbS chloroplast photosystem II 22 kDa component, psaN photosystem I reaction center subunit, psbY photosystem II core complex proteins, psbP photosystem II oxygen-evolving enhancer protein 2, petC Rieske FeS precursor protein 2, atpG gamma subunit of ATP synthase, petE plastocyanin A, psb27 photosystem II Psb27 protein, psbO chloroplast PsbO4 precursor, psaH photosystem I reaction centre subunit, psaG photosystem I reaction center V, petF ferredoxin, atpF ATP synthase subunit b, psbW photosystem II PsbW protein, petH ferredoxin-NADP reductase, psbQ photosystem II oxygen-evolving enhancer protein 3, psbR photosystem II 10 kDa polypeptide, psbA photosystem II protein D1

Fig. 5

H₂O₂ levels and production rate of O₂^·− in WT and transgenic plants. Data are expressed as the mean ± SE (n = 5, five biological replicates per lines). Different letters indicate statistically significant differences at P ≤ 0.05. a represents the content of H₂O₂. b indicates production rate of oxygen free radical. c shows histochemical staining of H₂O₂ and O₂^·−

Fig. 6

Activity and expression analysis of antioxidant enzymes in the WT, T3-1, T3-2, and T3-3 tobacco plants. A and D represent ascorbate peroxidase (APX), B and E represent catalase (CAT), and C and F represent superoxide dismutase (SOD). 18S RNA (GenBank Accession Number: AJ236016) was used as a housekeeping gene. The gene names and primers used for qRT-PCR analysis are presented in Additional file 6. Five biological replicates were used for each line to study of antioxidant enzyme activity. The standard error of the mean of three biological replicates (nested within three technical replicates) is represented by the error bars in qRT-PCR analysis

Fig. 7

Enhanced sensitivity of transgenic plants to oxidative stress. The 3-week-old plants were treated with 70 μM DCMU for 10 days

Fig. 8

Expression analysis of senescence marker genes in WT and transgenic plants. NtCP1 cysteine protease, SAG12 cysteine protease, Ntdin a tobacco senescence-associated gene, CHL chlorophyllase, GDH glutamate dehydrogenase, Nia nitrate reductase, GS2 chloroplastic glutamine synthetase, RBCS2B RuBisCO small subunit, CAB chlorophyll a/b binding protein. 18S RNA (GenBank Accession Number: AJ236016) was used as housekeeping gene. The gene names and primers used for qRT-PCR analysis are shown in Additional file 6. The standard error of the mean of three biological replicates (nested within three technical replicates) is represented by the error bars in qRT-PCR analysis