Multiple biological processes involved in the regulation of salicylic acid in Arabidopsis response to NO2 exposure
Abstract
Salicylic acid (SA) has been extensively implicated in plant response to abiotic stresses. However, just a few studies addressed the regulatory role of SA in plants exposed to NO2. In this study, we used Arabidopsis wild type (WT) and its SA-related mutants, including snc1 (suppressor of npr1-1, constitutive 1) with high endogenous SA, transgenic line nahG with low SA level, and npr1-1 (nonexpressor of PR gene) with SA signaling blockage to explore the possible regulation mechanism of SA, focusing on NO2-induced gene expression patterns. Plants were fumigated with 10 ppm NO2 for 8 h. The results showed that NO2 fumigation led to oxidative stress damage to all the tested plants as indicated by productions of H2O2, superoxide anion (O2
−) and malondialdehyde, and cell death shown by electrolyte leakage and Evans Blue staining. However, snc1 plants were injured to a much less degree, whereas nahG and npr1-1 plants to a larger degree as compared with WT plants. Correspondingly, the activities of superoxide dismutase, peroxidase, catalase and nitrate reductase were induced by NO2 fumigation to a higher extent in snc1 plants, and a less extent in nahG and npr1-1 plants relative to WT plants. The expressions of the genes related to antioxidative defense, disulfide bond reduction, SA-responsive and –biosynthesis pathway, flavonoid biosynthesis, nitrate metabolism and kinase were analyzed. The results showed that these genes were differentially expressed among the tested genotypes under NO2 fumigation, wherein the most were significantly up-regulated in snc1 plants relative to other tested genotypes. Based on the gene expression patterns, it was proposed that SA-mediated plant tolerance to NO2 stress was associated with multiple biological processes.
Introduction
Nitrogen dioxide (NO2) enters plant leaves mainly through stomata, and quickly dissolves in the aqueous environment of cell to form nitrate and nitrite (Nouchi, 2002). In low concentrations (normally ppb levels), NO2 not only is a nitrogen source for plant growth, but also as a vitalization signal to increase shoot size and the contents of cell constituents (Takahashi et al., 2005). However, exposure to high concentrations of NO2 (ppm levels) can cause a visible injury to plants. For instance, Arabidopsis leaves showed visible injury symptoms after 1 h exposure to 20 ppm of NO2, while 30 ppm caused complete leaf collapse and rapid wilting (Kasten et al., 2016). Our earlier study showed that adverse symptoms such as growth retardation, oxidative stress occurred in Arabidopsis plants exposed to 1 ppm NO2 for 3 h per day for 2 weeks (Xu et al., 2010). The accumulation of nitrite is thought to be a mediator of NO2-induced plant damage (Shimazaki et al., 1992; Kasten et al., 2016). Therefore, the reduction of nitrate and nitrite should be a detoxification way during plant response to NO2 exposure. In Arabidopsis, two genes NIA1 and NIA2 encoding nitrate reductase have been cloned. Analyses on the expression of NIA1 and NIA2 might provide an insight into plant tolerance to NO2 stress. The regulatory role of salicylic acid (SA) has been extensively investigated in plant responses to abiotic stresses (Khan et al., 2015; Bali et al., 2017) and especially to pathogen attack (Yan and Dong, 2014; Breen et al., 2017). However, just a few studies addressed the regulatory role of SA in plants exposed to NO2 (Xu et al., 2010; Kasten et al., 2016). The Arabidopsis sid2 and transgenic line nahG (both with SA deficiency) and npr1 (with SA signal blockage) exhibited more sensitive response than wild type plants to NO2 stress (Kasten et al., 2016), whereas the mutation with high accumulation capacity of SA enhanced plant tolerance to NO2 (Xu et al., 2010). However, the molecular mechanisms remain to be elucidated. Recently, we performed proteomic analyses on SA affecting plant response to NO2 stress, and found that endogenous SA level or signaling was involved in the regulation of a wide range of biological processes during NO2 exposure (unpublished data). This promoted us to further dissect the expression of related genes, with an aim to elucidate the biological processes associated with the SA-mediated plant tolerance to NO2.
The selected genes in this study are related to antioxidative defense, including three superoxide dismutase-encoding genes: CDS1, MSD1 and FSD1; four class III peroxidase-encoding genes: PRX2, PRX21, PRX27 and PRX42; three catalase-encoding genes: CAT1, CAT2 and CAT3, as well as ascorbate peroxidase 2 gene (APX2), monodehydroascorbate reductase 3 gene (MDAR3), glutathione reductase 1 gene (GR1), and alternative oxidase 2 gene (AOX2). Due to the significance of cellular redox status for the SA signaling involved in the reduction of disulfide bonds, several related genes were analyzed, such as those encoding protein disulfide isomerases 12 (PDI12), thioredoxins (TRX3 and TRXS) and NADPH-dependent thioredoxin reductase B (NTRB). We also detected the NO2-induced expression changes of SA-responsive or SA biosynthesis-related genes, such as pathogenesis-related genes (PR1 and PR2), phenylalanine ammonia lyase gene (PAL4). In addition, CHS, encoding chalcone synthase catalyzing the first step of flavonoid biosynthesis, and CRK19, encoding a member of cysteine-rich receptor-like protein kinases, were included in this study.
NPR1 is an important signaling component known to function downstream of SA, and an essential regulator of SA-mediated systemic acquired resistance (SAR) (Cao et al., 1997). The mutant npr1 loses both pathogenesis-related (PR) gene expression and SAR induced by avirulent pathogens or SAR-inducing agents such as SA (Glazebrook et al., 1996). In unchallenged conditions, NPR1 stays in the cytoplasm as an inactive oligomer linked through intermolecular disulfide bridges. However, upon challenges such as pathogens, SA increases the cellular redox state, leading to reduction of the disulfide bonds in NPR1 to a monomer, which enters the nucleus activating the expression of SA-responsive PR genes (Mou et al., 2003). In Arabidopsis, TRXs catalyze NPR1 oligomer reduction, and a mutation in TRX compromised NPR1-mediated disease resistance (Tada et al., 2008). TRXs are small thiol:disulfide oxidoreductases. In their reduced state, TRXs are able to reduce the disulfide bridges of numerous target proteins. Subsequently, the oxidized thioredoxins are reduced by thioredoxin reductases, forming together the so-called thioredoxin system. In Arabidopsis, there are three NTRs, namely NTRA (located in cytosol), NTRB (in mitochondria) and NTRC (in chloroplast), involving in thioredoxin reduction (Reichheld et al., 2005). The protein disulfide isomerases (PDIs) are capable of catalyzing the formation, breakage, or rearrangement of disulfide bonds in a wide range of substrate proteins (Yuen et al., 2016). PDI12 is a classic PDI-C isoform.
Arabidopsis SA high-accumulating mutant snc1 (suppressor of npr1-1, constitutive), SA-deficiency transgenic line nahG (naphthalene hydroxylase G), and SA-signal blockage mutant npr1-1 (nonexpressor of pathogenesis-related gene) were used in this study. The snc1 is isolated from EMS-mutagenized M2 progeny on the basis of the suppression of npr1-1 phenotype. This mutant has a high level of SA, which is required for manifestation of the specific phenotype under stressed conditions such as pathogen infection (Li et al., 2001). We found that the expression of most of the selected genes were dramatically induced by NO2 fumigation in snc1 plants relative to WT and other two mutant plants, suggesting that the proteins encoded by these genes might be involved in SA regulation in plant tolerance to NO2 stress.
Section snippets
Plant material and treatment
Seeds of wild type (WT) of Arabidopsis thaliana (L.) Heydn. (ecotype Columbia) and its mutants snc1 (Li et al., 2001), npr1-1 (Cao et al., 1994), and transgenic line nahG (Gaffney et al., 1993) were kindly given by Prof. Dong (Duke University). The seeds were sterilized in a 5% (v/v) sodium hypochlorite solution for 15 min, followed by three washes with sterile distilled water, then vernalized for 2 d at 4 °C in the dark. The seeds were sown in pots containing a mixture of
NO2-induced oxidative stress, antioxidative enzymes and nitrate reductase activity
Nitrogen dioxide (NO2) fumigation increased the contents of H2O2 by 128%, 38%, 167% and 139%, malondialdehyde (MDA) by 92%, 27%, 100% and 117%, and electrolyte leakage by 95%, 35%, 125% and 136%, respectively, in WT, snc1, nahG and npr1-1 plants relative to their respective non-fumigated plants (Table 2). These data demonstrated that NO2 fumigation caused oxidative stress damages to all the tested plants, but to a much less extent in snc1 plants, whereas a higher extent in nahG and npr1-1
Discussion
Based on the oxidative stress indicators (Fig. 1 and Table 2), this study showed that snc1 plants (with high endogenous SA) had a much stronger tolerance to NO2 stress, whereas nahG plants (low SA) and npr1-1 plants (blockage in SA signaling) exhibited more sensitivity, as compared with WT plants. This was agreed with the results obtained in a long-term exposure experiment where plants were fumigated with 1 ppm NO2 for 3 h per day for 14 days (Xu et al., 2010). A recent study also showed that
Conflict of interest
The authors declare that they have no conflict of interest.
Funding
This research was supported by the National Natural Science Foundation of China (Grant No. 31570502 to LGZ, 31572213 and 31270446 to HL).
Author contributions
HL and LGZ designed and carried out the research. QY, WYY, YQS, HLL and JYG contributed to carry out the physiological, biochemical and the qRT-PCR analyses. HL and LGZ analyzed the data and wrote the manuscript. All authors read and approved the manuscript.
Acknowledgements
The authors acknowledge X. Dong for Arabidopsis seeds. We thank Dr. Y. Han for reading the manuscript.
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