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Article

Knockdown of Quinolinate Phosphoribosyltransferase Results in Decreased Salicylic Acid-Mediated Pathogen Resistance in Arabidopsis thaliana

by
Shengchun Li
*,
Haiyan Ding
,
Yi Deng
and
Jiang Zhang
State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(16), 8484; https://doi.org/10.3390/ijms22168484
Submission received: 30 March 2021 / Revised: 28 July 2021 / Accepted: 3 August 2021 / Published: 6 August 2021
(This article belongs to the Section Molecular Plant Sciences)
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Abstract

:
Nicotinamide adenine dinucleotide (NAD) is a pivotal coenzyme that has emerged as a central hub linking redox equilibrium and signal transduction in living cells. The homeostasis of NAD is required for plant growth, development, and adaption to environmental stresses. Quinolinate phosphoribosyltransferase (QPRT) is a key enzyme in NAD de novo synthesis pathway. T-DNA-based disruption of QPRT gene is embryo lethal in Arabidopsis thaliana. Therefore, to investigate the function of QPRT in Arabidopsis, we generated transgenic plants with decreased QPRT using the RNA interference approach. While interference of QPRT gene led to an impairment of NAD biosynthesis, the QPRT RNAi plants did not display distinguishable phenotypes under the optimal condition in comparison with wild-type plants. Intriguingly, they exhibited enhanced sensitivity to an avirulent strain of Pseudomonas syringae pv. tomato (Pst-avrRpt2), which was accompanied by a reduction in salicylic acid (SA) accumulation and down-regulation of pathogenesis-related genes expression as compared with the wild type. Moreover, oxidative stress marker genes including GSTU24, OXI1, AOX1 and FER1 were markedly repressed in the QPRT RNAi plants. Taken together, these data emphasized the importance of QPRT in NAD biosynthesis and immunity defense, suggesting that decreased antibacterial immunity through the alteration of NAD status could be attributed to SA- and reactive oxygen species-dependent pathways.

1. Introduction

Pyridine nucleotide co-enzymes are ubiquitous in living organisms [1]. Nicotinamide adenine dinucleotide (NAD) and its phosphate derivative NADP, exist in oxidized forms (NAD+ or NADP+, respectively) or reduced forms (NADH or NADPH, respectively). They mediate a wide range of redox reactions, and thus impact virtually all metabolic reactions in the cell [2]. In addition to their functional roles in redox regulation, NADs also plays important roles in cell signaling, including intracellular Ca2+ signalling via NAD-derived cyclic ADP-ribose [3], translational modification of target proteins by the transfer of ADP-ribose from NAD catalyzed by poly(ADP-ribose) polymerases and mono(ADP-ribosyl)transferase [4], and epigenetic regulations by the sirtuin histone deacetylases [5,6].
In plants, NAD has also been documented to act as a signal in response to environmental stresses, including pathogen infections [7,8,9]. For instance, quinolinate-induced stimulation of intracellular NAD in Arabidopsis expressing nadC gene from Escherichia coli, which encodes quinolinate phosphoribosyltransferase (QPRT), enhances defense gene expression and resistance to diverse bacterial and fungal pathogens [10,11]. Overexpression of Arabidopsis Nudix (nucleoside diphosphates linked to some moiety X) hydrolase gene, hydrolase homolog 6 (AtNUDT6), encoding an ADP-ribose/NADH pyrophosphohydrolase, and disruption of AtNUDT6 [12], AtNUDT7 [13,14], or AtNUDT8 [15] all lead to changes in intracellular NADH levels and salicylic acid (SA)-mediated immune signaling. Additionally, exogenous NAD(P) treatment in Arabidopsis can induce SA-dependent and -independent expression of pathogenesis-related (PR) genes and resistance to bacterial pathogens [16,17]. The recent identification of plasma membrane-localized NAD(P) receptors LecRK-I.8 and LecRK-VI.2 confirmed that NAD(P) act as extracellular signals [18,19].
NAD can be synthesized via both a de novo pathway and a salvage pathway in plants (Figure 1) [2,9,20]. The de novo pathway starts in plastids using aspartate as precursor. In plastids, quinolinate is produced from aspartate and dihydroxyacetone phosphate by aspartate oxidase (AO) plus quinolinate synthase (QS), and quinolinate is rapidly converted to nicotinate mononucleotide (NaMN) by QPRT. NaMN is thereafter converted to NAD in the cytosol through adenylation by NaMN adenylyltransferase (NaMNAT) and amidation by NAD synthase (NADS). AO, QS, QPRT and NaMNAT are single genes in the Arabidopsis genome. The T-DNA insertion of either of these genes exhibited developmental defects or was embryo lethal [20,21], indicating an essential role of the de novo pathway in plant growth and development. NaMNAT and NADS also participate in the NAD salvage pathway (Figure 1) [9]. Unlike the disrupted AO allele [20], AO-knockdown mutants are viable and fertile plants, but harbor impaired stomatal immunity against a coronatine-deficient strain of Pseudomonas syringae pv. tomato (Pst) DC3000 [22]. Arabidopsis QS consists of a quinolinate synthase (NadA) and a SufE3 domain required for incorporation of the Fe-S cluster [23]. A non-lethal mutation in the SufE domain is responsible for the early senescence phenotype of the onset of leaf death5 (old5) mutant [24]. Rather than decreased NAD, this effect was associated with increased level of NAD, which was attributed to enhanced activities of the salvage pathway in the old5 mutant [24]. Recent studies have shown that a point mutation (Q288E substitution) in the region encoding the NadA domain of the QS gene resulted in markedly decreased levels of NAD, and caused salt and abscisic acid (ABA) hypersensitivity in Arabidopsis [25,26]. To date, no detailed studies of QPRT knockdown mutants of Arabidopsis have been reported.
The NAD salvage pathway starts from nicotinamide (Figure 1), which is sequentially catalyzed by nicotinamidase (NIC), nicotinate phosphoribosyltransferase (NaPRT), NaMNAT, and NADS. Three NIC genes (NIC1, NIC2, and NIC3) have been identified in Arabidopsis [27,28]. The major NIC appears to be NIC1, and null mutant nic1-1 shows decreased pools of pyridine nucleotides in many tissues and is hypersensitive to salt and ABA treatments, pointing to a role for the pathway in recycling and maintaining NAD pools [27]. Moreover, a 60-fold increase in nicotinamide contents in nic1-1 Arabidopsis mutants was shown to inhibit aphid reproductive potential [29]. The NIC2 gene is most strongly expressed in mature seeds. Increased levels of NAD was observed in nic2-1 mutant, and this was associated with increased seed dormancy [28].
Since disruption of QPRT causes lethality in Arabidopsis [20], QPRT knockdown RNAi lines were generated to assess the effects of a reduced level of QPRT on pathogen infection. Decreased NAD levels in the QPRT RNAi plants led to enhanced pathogen sensitivity accompanied by decreased expression of PR genes and oxidative marker genes, and also reduced SA accumulation.

2. Results

2.1. Generation of Transgenic Plants with Decreased QPRT Expression

To assess the effects of decreased expression of QPRT, we generated RNAi knockdown lines by transforming wild-type Arabidopsis plants with a 343 bp fragment from cDNA in sense and antisense directions under the 35S promoter (Figure 2A). We selected two independent RNAi lines (At-pHY6#2 and At-pHY6#13) with greatly reduced QPRT mRNA levels (Figure 2B) and used the homologous T3 progeny of these lines in subsequent analyses (Figure S1). As expected, the QPRT RNAi lines showed reduced levels of NAD (Figure 2C). Moreover, a significant decrease in NADP content was observed in At-pHY6#13 plants (Figure 2D). Neither the NADH nor the NADPH pool was significantly affected in the RNAi lines (Figure 2C,D). However, reduced QPRT expression did not cause aberrant phenotypes when plants were grown under the conditions of these experiments (Figure S2).

2.2. Decreased QPRT Enhances Sensitivity to Pst-avrRpt2

To determine whether decreased expression of QPRT affects plant resistance to biotic stress, we investigated the response of the RNAi lines to an avirulent pathogen of Pst DC3000 harboring the elicitor avrRpt2 (Pst-avrRpt2). As shown Figure 3A, the growth of Pst-avrRpt2 in the QPRT RNAi plants was significantly higher than that in the wild-type plants, both at 24 h post inoculation (24 hpi) and 48 hpi. To understand the mechanism by which the decreased QPRT expression led to enhanced disease sensitivity, we examined defense responses including transcripts of a key gene involved in SA synthesis (isochorismate synthase 1, ICS1) and PR genes, as well as SA accumulation. Quantitative RT-PCR analysis showed that transcripts of ICS1, PR1 and PR5 were reduced in response to Pst-avrRpt2 in the QPRT RNAi plants in comparison with Col-0 at 48 hpi (Figure 3B–D). Moreover, down-regulation of these transcriptional levels coincided with reduced accumulation of total SA in the RNAi lines (Figure 3E).

2.3. Decreased QPRT Affects Redox Homeostasis

To test whether the observed sensitivity was accompanied by changes in NAD pools, we determined the expression level of QPRT gene and contents of pyridine nucleotides in the QPRT RNAi plants infected by Pst-avrRpt2. There was a relatively low level of QPRT expression in the RNAi lines in comparison with the wild-type plants at 48 hpi (Figure S3). Consistent with QPRT expression, the NAD levels were lower in the QPRT RNAi plants than that in Col-0 at 48 hpi, whereas much higher levels of NADH were seen in the RNAi lines (Figure 4A,B). In addition, the levels of NADP and NADPH did not differ significantly between the RNAi lines and Col-0 at 48 hpi (Figure 4C,D).
Reactive oxygen species (ROS) burst has been well established as an integral aspect of plant immunity [30,31], and NAD has been known to stimulate ROS production [11]. To further investigate overall cellular redox states, we measured changes in hydrogen peroxide (H2O2) indirectly by measuring changes in the transcript levels of four H2O2 marker genes, cytosolic glutathione S-transferase TAU 24 (GSTU24, AT1G17170) [32] and oxidative signal-inducible 1 (OXI1, AT3G25250) [33], mitochondrial alternative oxidase 1 (AOX1, AT3G22370) [34], and chloroplast ferritin 1 (FER1, AT5G01600) [35]. The expression of these genes was reduced in the RNAi plants in comparison with wild-type plants at 48 hpi (Figure 5).

3. Discussion

Because the homozygous null alleles of qprt mutants of Arabidopsis are embryo-lethal [20], QPRT RNAi plants were generated to assess the effects of constitutively reduced level of QPRT. The amount of NAD was lower in the QPRT RNAi plants, while the NADH levels were similar to those observed in control plants under optimal conditions (Figure 2C). Pyridine nucleotide measurements in this study represent total nucleotide pools, i.e., they include free pools and those bound to proteins. It was revealed that the free NADH level can be maintained at a more constant value than oxidized NAD and total NAD [36], partially providing an explanation for the NADH levels not changing significantly in the RNAi lines. No obvious phenotype was observed in the RNAi lines under the conditions of these experiments (Figure S2).
A growing body of evidence suggests that NAD plays a crucial role in plant immunity [10,11,16,22,37]. Notably, overexpression of the bacterial NAD biosynthesis gene nadC, which increased intracellular NAD levels with the addition of quinolinate, resulted in heightened resistance to avirulent pathogen Pst-AvrRpm1, but not to virulent strain Pst DC3000 [10]. In the current study, we tested whether a deficient NAD synthesis pathway in QPRT RNAi lines would have increased susceptibility to an avirulent bacterial strain. As expected, after infection with the avirulent avrRpt2-containing Pst DC3000 strain (Pst-avrRpt2), the QPRT RNAi plants exhibited increased bacterial proliferation compared with Col-0. The enhanced sensitivity to Pst-avrRpt2 was associated with suppressed expression of the defense-related genes (ICS1, PR1 and PR5), and this was consistent with decreased SA accumulation (Figure 3). In addition, the NAD levels were decreased following the infection with Pst-avrRpt2, whereas the NADH levels were increased (Figure 2C; Figure 4A,B). A much higher level of NADH was observed in the RNAi lines in comparison with Col-0 after Pst-avrRpt2 infection. The observed alterations of NAD(H) pools led to a lower NAD/NADH ratio in the RNAi lines than that in wild-type plants. Indeed, the physiologically relevant ratio of NAD to NADH is generally high, favoring hydride transfer from a substrate to NAD to form NADH in plant cells [38]. The results of the current study support that QPRT enzyme is critical for the steady state of NAD and the homeostasis of NAD/NADH in Arabidopsis.
The cytosolic GSTU24 gene is known to be H2O2 inducible [32,39] and up-regulated by Pst-avrRpt2 [40,41]. OXI1, a serine/threonine kinase, activates the MAPK3/6 cascade in an H2O2-dependent manner, which has been implicated in some pathogen responses and root hair development [33,42]. AOX1 encodes a mitochondrial alternative oxidase that has been shown to be induced by Pst attack [43] and links mitochondrial ROS to cell death [44,45]. FER1, encoding chloroplast-localized ferritins, has been shown to play an important role in keeping Fe2+ levels at a minimum upon increased intracellular H2O2 [35,46]. In the current study, reduced expression of four H2O2 marker genes suggests redox perturbations occurred in different subcellular compartments in QPRT RNAi plants. Given that the cytosol is the site of the final step of NAD biosynthesis (Figure 1), specific transport proteins are required to shuttle NAD across intracellular membranes for organellar import of NAD into chloroplasts, mitochondria and peroxisomes [47,48,49,50,51]. NAD transport could influence cellular redox balance, thus may lead to redox perturbations in different subcellular compartments of QPRT RNAi plants.
Collectively, these results indicate that QPRT is important in NAD biosynthesis, and contributes to plant resistance through SA- and ROS-dependent pathways. Additionally, the availability of the QPRT RNAi lines allow future detailed studies on the role of NAD metabolism in many aspects of plant biology including, but not limited to, immunity, development, signaling, and biosynthesis [8,9,38].

4. Materials and Methods

4.1. Plant Materials and Growth Condition

The RNAi lines used in this study were derived from the wild-type Arabidopsis thaliana Columbia (Col-0) ecotype. Plants were cultivated in controlled growth chambers in a photoperiod of 8 h light/16 h dark, an irradiance of 100 μmol·m−2s−1 at leaf level, at temperatures of 22 °C day/20 °C night and 75–80% humidity.

4.2. Generation of RNAi Plants

To obtain RNAi plants, partial coding region for QPRT (AT2G01350) was amplified with RiQPRT-Fwd1/RiQPRT-Rev1 and RiQPRT-Fwd2/RiQPRT-Rev2 primers (Table S1). The primers harbor a short homologous sequence (underlined) to pHELLSGATE8 vector. Two QPRT PCR products were then cloned into the pHELLSGATE8 vector via homologous recombination between the XhoI-XhoI sites in the sense orientation and the XbaI-XbaI sites in the antisense orientation to generate RNAi construct pHY6 (Figure 2A) [52]. The construct was transformed into the Agrobacterium tumefaciens GV3101 strain, which was then used to transform Arabidopsis Col-0 using the floral dip method [53]. Transgenic plants were selected on Murashige and Skoog (MS) media containing 50mg·L−1 kanamycin. Single insertion lines were selected by 3:1 segregation on selection media.

4.3. Pathogen Test

The avirulence strain of Pseudomonas syringae pv. tomato DC3000 carrying the avirulent gene avrRpt2 (Pst-avrRpt2; a gift from Prof. Jianmin Zhou, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) was used for resistance tests with a medium titer of 5 × 105 colony-forming units (cfu) mL−1. Whole leaves of 5-week-old plants were infiltrated with a 1-mL needleless syringe in the middle of the photoperiod. Six to eight leaves were made by pooling two leaf discs (φ = 0.45 cm each) harvested from different inoculated leaves of different plants. Bacterial growth was examined by grinding leaf discs in 400 mL of water, plating appropriate dilutions on solid King’s B medium with 100 mg·L−1 rifampicin and 25 mg·L−1 kanamycin, and quantifying colony numbers. Bacterial cfu were counted at 0, 24 and 48 hpi. Experiments were repeated three times with similar results.

4.4. RNA Isolation and Quantitative Reverse Transcription-PCR

Total RNA was isolated from leaf samples using RNAiso Plus Reagent (Takara, Dalian, China) according to the manufacturer’s instructions. Hifair® Ⅱ 1st Strand cDNA Synthesis Kit with gDNA digester (Yeasen, China) was used to synthesize cDNA. Quantitative reverse transcription-PCR (qRT-PCR) was performed on Bio-Rad CFX Connect Real-Time System (Bio-Rad, USA) using the SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) kit (Takara, Dalian, China). The ACTIN2 gene was used as an internal control to normalize for specific gene expression in the samples. Each treatment was replicated with at least three independent biological sample preparations. Quantitative analysis of gene expression was performed using the 2−ΔΔCT method [54]. Gene primers are listed in Table S1.

4.5. Metabolite Measurements

Reduced and oxidized forms of NAD and NADP were determined by spectrophotometric assays, as previously described [55,56]. Total SA concentration was measured by high-performance liquid chromatography-fluorescence according to Langlois-Meurinne et al. (2005) [57].

4.6. Statistical Analysis

Statistical significance was determined using the Student’s t-test. A p value of <0.05 was considered significant and indicated by an asterisk in the Figures.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/ijms22168484/s1. Figure S1. Seed assays confirming the homozygous status of the QPRT RNAi plants. Figure S2. Knockdown of QPRT expression by RNAi did not affect plant growth under optimal growth condition. Figure S3 QPRT mRNA levels at 48 hpi. Table S1. List of primers used in the study.

Author Contributions

Conceptualization, S.L.; methodology, H.D., Y.D., writing—original draft preparation, H.D. and Y.D.; writing—review and editing, J.Z. and S.L.; funding acquisition, S.L. All authors have read and agreed to the published version of the manuscript.

Funding

S.L. was supported by the National Natural Science Foundation of China (32071477 and 31700227) and Innovation Base for Introducing Talents of Discipline of Hubei Province (2019BJH021).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

This study did not report any data.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Noctor, G.; Hager, J.; Li, S. Biosynthesis of NAD and its manipulation in plants. Adv. Bot 2011, 58, 153–201. [Google Scholar] [CrossRef]
  2. Noctor, G.; Queval, G.; Gakière, B. NAD(P) synthesis and pyridine nucleotide cycling in plants and their potential importance in stress conditions. J. Exp. Bot. 2006, 57, 1603–1620. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Sánchez, J.; Duque, P.; Chua, N. ABA activates ADPR cyclase and cADPR induces a subset of ABA-responsive genes in Arabidopsis. Plant J. 2004, 38, 381–395. [Google Scholar] [CrossRef] [PubMed]
  4. Briggs, A.G.; Bent, A.F. Poly(ADP-ribosyl)ation in plants. Trends Plant Sci. 2011, 16, 372–380. [Google Scholar] [CrossRef]
  5. Kupis, W.; Palyga, J.; Tomal, E.; Niewiadomska, E. The role of sirtuins in cellular homeostasis. J. Physiol Biochem 2016, 72, 371–380. [Google Scholar] [CrossRef] [Green Version]
  6. Imai, S.; Armstrong, C.; Kaeberlein, M.; Guarente, L. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 2000, 403, 795–800. [Google Scholar] [CrossRef]
  7. Berger, F.; Ramirez-Hernandez, M.H.; Ziegler, M. The new life of a centenarian: Signalling functions of NAD(P). Trends Biochem. Sci. 2004, 29, 111–118. [Google Scholar] [CrossRef]
  8. Gakière, B.; Fernie, A.R.; Petriacq, P. More to NAD(+) than meets the eye: A regulator of metabolic pools and gene expression in Arabidopsis. Free Radic. Bio. Med. 2018, 122, 86–95. [Google Scholar] [CrossRef]
  9. Gakière, B.; Hao, J.; de Bont, L.; Pétriacq, P. NAD biosynthesis and signaling in plants. Crit. Rev. Plant Sci. 2018, 37, 1–49. [Google Scholar] [CrossRef]
  10. Pétriacq, P.; de Bont, L.; Hager, J.; Didierlaurent, L.; Mauve, C.; Guérard, F.; Noctor, G.; Pelletier, S.; Renou, J.; Tcherkez, G.; et al. Inducible NAD overproduction in Arabidopsis alters metabolic pools and gene expression correlated with increased salicylate content and resistance to Pst-AvrRpm1. Plant J. 2012, 70, 650–665. [Google Scholar] [CrossRef] [PubMed]
  11. Pétriacq, P.; Ton, J.; Patrit, O.; Tcherkez, G.; Gakiere, B. NAD acts as an integral regulator of multiple defense layers. Plant Physiol. 2016, 172, 1465–1479. [Google Scholar] [CrossRef] [Green Version]
  12. Ishikawa, K.; Yoshimura, K.; Harada, K.; Fukusaki, E.; Ogawa, T.; Tamoi, M.; Shigeoka, S. AtNUDX6, an ADP-ribose/NADH pyrophosphohydrolase in Arabidopsis, positively regulates NPR1-dependent salicylic acid signaling. Plant Physiol. 2010, 152, 2000–2012. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Ge, X.; Li, G.J.; Wang, S.B.; Zhu, H.; Zhu, T.; Wang, X.; Xia, Y. AtNUDT7, a negative regulator of basal immunity in Arabidopsis, modulates two distinct defense response pathways and is involved in maintaining redox homeostasis. Plant Physiol. 2007, 145, 204–215. [Google Scholar] [CrossRef] [Green Version]
  14. Bartsch, M.; Gobbato, E.; Bednarek, P.; Debey, S.; Schultze, J.L.; Bautor, J.; Parker, J.E. Salicylic acid-independent enhanced disease susceptibility1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenase FMO1 and the Nudix hydrolase NUDT7. Plant Cell 2006, 18, 1038–1051. [Google Scholar] [CrossRef] [Green Version]
  15. Fonseca, J.P.; Dong, X. Functional characterization of a Nudix hydrolase AtNUDX8 upon pathogen attack indicates a positive role in plant immune responses. PLoS ONE 2014, 9, e114119. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Zhang, X.; Mou, Z. Extracellular pyridine nucleotides induce PR gene expression and disease resistance in Arabidopsis. Plant J. 2009, 57, 302–312. [Google Scholar] [CrossRef] [PubMed]
  17. Wang, C.; Zhang, X.; Mou, Z. Comparison of nicotinamide adenine dinucleotide phosphate-induced immune responses against biotrophic and necrotrophic pathogens in Arabidopsis thaliana. Plant Signal. Behav. 2016, 11, e1169358. [Google Scholar] [CrossRef] [PubMed]
  18. Wang, C.; Zhou, M.; Zhang, X.; Yao, J.; Zhang, Y.; Mou, Z. A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana. Elife 2017, 6, e25474. [Google Scholar] [CrossRef]
  19. Wang, C.; Huang, X.; Li, Q.; Zhang, Y.; Li, J.L.; Mou, Z. Extracellular pyridine nucleotides trigger plant systemic immunity through a lectin receptor kinase/BAK1 complex. Nat. Commun. 2019, 10, 4810. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  20. Katoh, A.; Uenohara, K.; Akita, M.; Hashimoto, T. Early steps in the biosynthesis of NAD in Arabidopsis start with aspartate and occur in the plastid. Plant Physiol. 2006, 141, 851–857. [Google Scholar] [CrossRef] [Green Version]
  21. Hashida, S.N.; Takahashi, H.; Kawai-Yamada, M.; Uchimiya, H. Arabidopsis thaliana nicotinate/nicotinamide mononucleotide adenyltransferase (AtNMNAT) is required for pollen tube growth. Plant J. 2007, 49, 694–703. [Google Scholar] [CrossRef] [PubMed]
  22. Macho, A.; Boutrot, F.; Rathjen, J.; Zipfel, C. Aspartate oxidase plays an important role in Arabidopsis stomatal immunity. Plant Physiol. 2012, 159, 1845–1856. [Google Scholar] [CrossRef] [Green Version]
  23. Murthy, U.M.N.; Ollagnier-de-Choudens, S.; Sanakis, Y.; Abdel-Ghany, S.E.; Rousset, C.; Ye, H.; Fontecave, M.; Pilon-Smits, E.A.H.; Pilon, M. Characterization of Arabidopsis thaliana SufE2 and SufE3: Functions in chloroplast iron-sulfur cluster assembly and Nad synthesis. J. Biol. Chem. 2007, 282, 18254–18264. [Google Scholar] [CrossRef] [Green Version]
  24. Schippers, J.H.; Nunes-Nesi, A.; Apetrei, R.; Hille, J.; Fernie, A.R.; Dijkwel, P.P. The Arabidopsis onset of leaf death5 mutation of quinolinate synthase affects nicotinamide adenine dinucleotide biosynthesis and causes early ageing. Plant Cell 2008, 20, 2909–2925. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Wei, M.; Zhuang, Y.; Li, H.; Li, P.; Huo, H.; Shu, D.; Huang, W.; Wang, S. The cloning and characterization of hypersensitive to salt stress mutant, affected in quinolinate synthase, highlights the involvement of NAD in stress-induced accumulation of ABA and proline. Plant J. 2020, 102, 85–98. [Google Scholar] [CrossRef]
  26. Hong, Y.; Wang, Z.; Shi, H.; Yao, J.; Liu, X.; Wang, F.; Zeng, L.; Xie, Z.; Zhu, J.K. Reciprocal regulation between nicotinamide adenine dinucleotide metabolism and abscisic acid and stress response pathways in Arabidopsis. PLoS Genet. 2020, 16, e1008892. [Google Scholar] [CrossRef]
  27. Wang, G.; Pichersky, E. Nicotinamidase participates in the salvage pathway of NAD biosynthesis in Arabidopsis. Plant J. 2007, 49, 1020–1029. [Google Scholar] [CrossRef]
  28. Hunt, L.; Holdsworth, M.J.; Gray, J.E. Nicotinamidase activity is important for germination. Plant J. 2007, 51, 341–351. [Google Scholar] [CrossRef]
  29. Sattar, S.; Martinez, M.T.; Ruiz, A.F.; Hanna-Rose, W.; Thompson, G.A. Nicotinamide inhibits aphid fecundity and impacts survival. Sci. Rep. 2019, 9, 19709. [Google Scholar] [CrossRef] [PubMed]
  30. Castro, B.; Citterico, M.; Kimura, S.; Stevens, D.; Wrzaczek, M.; Gitta Coakerg, G. Stress-induced reactive oxygen species compartmentalization, perception and signalling. Nat. Plants 2021, 7, 403–412. [Google Scholar] [CrossRef]
  31. Torres, M.A. ROS in biotic interactions. Physiol. Plantarum 2010, 138, 414–429. [Google Scholar] [CrossRef]
  32. Chaouch, S.; Queval, G.; Noctor, G. AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis. Plant J. 2012, 69, 613–627. [Google Scholar] [CrossRef] [PubMed]
  33. Rentel, M.; Lecourieux, D.; Ouaked, F.; Usher, S.; Petersen, L.; Okamoto, H.; Knight, H.; Peck, S.; Grierson, C.; Hirt, H.; et al. OXI1 kinase is necessary for oxidative burst-mediated signalling in Arabidopsis. Nature 2004, 427, 858–861. [Google Scholar] [CrossRef] [PubMed]
  34. Vanlerberghe, G.C. Alternative oxidase: A mitochondrial respiratory pathway to maintain metabolic and signaling homeostasis during abiotic and biotic stress in plants. Int. J. Mol. Sci. 2013, 14, 6805–6847. [Google Scholar] [CrossRef] [PubMed]
  35. Petit, J.; Briat, J.; Lobréaux, S. Structure and differential expression of the four members of the Arabidopsis thaliana ferritin gene family. Biochem. J. 2001, 359, 575–582. [Google Scholar] [CrossRef]
  36. Kasimova, M.R.; Grigiene, J.; Krab, K.; Hagedorn, P.H.; Flyvbjerg, H.; Andersen, P.E.; Moller, I.M. The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions. Plant Cell 2006, 18, 688–698. [Google Scholar] [CrossRef] [Green Version]
  37. Zhang, X.; Mou, Z. Expression of the human NAD(P)-metabolizing ectoenzyme CD38 compromises systemic acquired resistance in Arabidopsis. Mol. Plant Microbe. Interact. 2012, 25, 1209–1218. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Smith, E.N.; Schwarzländer, M.; Ratcliffe, R.G.; Kruger, N.J. Shining a light on NAD- and NADP-based metabolism in plants. Trends Plant Sci. 2021. [Google Scholar] [CrossRef]
  39. Mhamdi, A.; Mauve, C.; Gouia, H.; Saindrenan, P.; Hodges, M.; Noctor, G. Cytosolic NADP-dependent isocitrate dehydrogenase contributes to redox homeostasis and the regulation of pathogen responses in Arabidopsis leaves. Plant Cell Environ. 2010, 33, 1112–1123. [Google Scholar] [CrossRef]
  40. De Vos, M.; Van Oosten, V.; Van Poecke, R.; Van Pelt, J.; Pozo, M.; Mueller, M.; Buchala, A.; Métraux, J.; Van Loon, L.; Dicke, M.; et al. Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Mol. Plant Microbe. Interact. 2005, 18, 923–937. [Google Scholar] [CrossRef] [Green Version]
  41. Gullner, G.; Komives, T.; Kiraly, L.; Schroder, P. Glutathione S-transferase enzymes in plant-pathogen interactions. Front. Plant Sci. 2018, 9, 1836. [Google Scholar] [CrossRef] [Green Version]
  42. Petersen, L.N.; Ingle, R.A.; Knight, M.R.; Denby, K.J. OXI1 protein kinase is required for plant immunity against Pseudomonas syringae in Arabidopsis. J. Exp. Bot. 2009, 60, 3727–3735. [Google Scholar] [CrossRef] [Green Version]
  43. Simons, B.; Millenaar, F.; Mulder, L.; Van Loon, L.; Lambers, H. Enhanced expression and activation of the alternative oxidase during infection of Arabidopsopsis with Pseudomonas syringae pv tomato. Plant Physiol. 1999, 120, 529–538. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Garmier, M.; Priault, P.; Vidal, G.; Driscoll, S.; Djebbar, R.; Boccara, M.; Mathieu, C.; Foyer, C.H.; De Paepe, R. Light and oxygen are not required for harpin-induced cell death. J. Biol. Chem. 2007, 282, 37556–37566. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Vidal, G.; Ribas-Carbo, M.; Garmier, M.; Dubertret, G.; Rasmusson, A.G.; Mathieu, C.; Foyer, C.H.; De Paepe, R. Lack of respiratory chain complex I impairs alternative oxidase engagement and modulates redox signaling during elicitor-induced cell death in tobacco. Plant Cell 2007, 19, 640–655. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Arnaud, N.; Murgia, I.; Boucherez, J.; Briat, J.F.; Cellier, F.; Gaymard, F. An iron-induced nitric oxide burst precedes ubiquitin-dependent protein degradation for Arabidopsis AtFer1 ferritin gene expression. J. Biol. Chem. 2006, 281, 23579–23588. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Palmieri, F.; Rieder, B.; Ventrella, A.; Blanco, E.; Do, P.T.; Nunes-Nesi, A.; Trauth, A.U.; Fiermonte, G.; Tjaden, J.; Agrimi, G.; et al. Molecular identification and functional characterization of Arabidopsis thaliana mitochondrial and chloroplastic NAD+ carrier proteins. J. Biol. Chem. 2009, 284, 31249–31259. [Google Scholar] [CrossRef] [Green Version]
  48. De Souza Chaves, I.; Feitosa-Araujo, E.; Florian, A.; Medeiros, D.B.; da Fonseca-Pereira, P.; Charton, L.; Heyneke, E.; Apfata, J.A.C.; Pires, M.V.; Mettler-Altmann, T.; et al. The mitochondrial NAD(+) transporter (NDT1) plays important roles in cellular NAD(+) homeostasis in Arabidopsis thaliana. Plant J. 2019, 100, 487–504. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  49. Luo, L.; He, Y.; Zhao, Y.; Xu, Q.; Wu, J.; Ma, H.; Guo, H.; Bai, L.; Zuo, J.; Zhou, J.M.; et al. Regulation of mitochondrial NAD pool via NAD(+) transporter 2 is essential for matrix NADH homeostasis and ROS production in Arabidopsis. Sci. China Life Sci. 2019, 62, 991–1002. [Google Scholar] [CrossRef]
  50. Van Roermund, C.W.; Schroers, M.G.; Wiese, J.; Facchinelli, F.; Kurz, S.; Wilkinson, S.; Charton, L.; Wanders, R.J.; Waterham, H.R.; Weber, A.P.; et al. The peroxisomal NAD carrier from Arabidopsis imports NAD in exchange with AMP. Plant Physiol. 2016, 171, 2127–2139. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  51. Bernhardt, K.; Wilkinson, S.; Weber, A.P.; Linka, N. A peroxisomal carrier delivers NAD(+) and contributes to optimal fatty acid degradation during storage oil mobilization. Plant J. 2012, 69, 1–13. [Google Scholar] [CrossRef] [PubMed]
  52. Wu, Y.Y.; You, L.L.; Li, S.C.; Ma, M.Q.; Wu, M.T.; Ma, L.X.; Bock, R.; Chang, L.; Zhang, J. In vivo assembly in Escherichia coli of transformation vectors for plastid genome engineering. Front. Plant Sci. 2017, 8, 1454. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Zhang, X.; Henriques, R.; Lin, S.S.; Niu, Q.W.; Chua, N.H. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat. Protoc. 2006, 1, 641–646. [Google Scholar] [CrossRef]
  54. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  55. Noctor, G.; Mhamdi, A.; Foyer, C.H. Oxidative stress and antioxidative systems: Recipes for successful data collection and interpretation. Plant Cell Environ. 2016, 39, 1140–1160. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Queval, G.; Noctor, G. A plate reader method for the measurement of NAD, NADP, glutathione, and ascorbate in tissue extracts: Application to redox proWling during Arabidopsis rosette development. Anal. Biochem 2007, 363, 58–69. [Google Scholar] [CrossRef] [PubMed]
  57. Langlois-Meurinne, M.; Gachon, C.M.; Saindrenan, P. Pathogen-responsive expression of glycosyltransferase genes UGT73B3 and UGT73B5 is necessary for resistance to Pseudomonas syringae pv tomato in Arabidopsis. Plant Physiol. 2005, 139, 1890–1901. [Google Scholar] [CrossRef] [PubMed] [Green Version]
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Figure 1. Schematic representation of NAD biosynthesis pathway in plants. The de novo biosynthesis of NAD starts from aspartate in the plastid. Abbreviations: AO, aspartate oxidase; NaAD, nicotinate adenine dinucleotide; NADK, NAD kinase; NADS, NAD synthetase; NaMN(AT), nicotinate mononucleotide (adenylyltransferase); NaPRT, nicotinate phosphoribosyltransferase; NIC, nicotinamidase; QPRT, quinolinate phosphoribosyltransferase; QS, quinolinate synthase.
Figure 1. Schematic representation of NAD biosynthesis pathway in plants. The de novo biosynthesis of NAD starts from aspartate in the plastid. Abbreviations: AO, aspartate oxidase; NaAD, nicotinate adenine dinucleotide; NADK, NAD kinase; NADS, NAD synthetase; NaMN(AT), nicotinate mononucleotide (adenylyltransferase); NaPRT, nicotinate phosphoribosyltransferase; NIC, nicotinamidase; QPRT, quinolinate phosphoribosyltransferase; QS, quinolinate synthase.
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Figure 2. Generation of QPRT RNAi lines and determination of pyridine nucleotides contents in Arabidopsis. (A) The RNAi construct pHY6 contained a 35S promoter, a sense fragment of QPRT cDNA from 164 to 506, the PDK intron, the QPRT fragment in antisense orientation, and an OCS terminator. (B) QPRT mRNA levels analyzed by qRT-PCR. Contents of NAD(H) (C) and NADP(H) (D) were quantified. White bars, reduced forms; black bars, oxidized forms. Data are means ± SE of three biological replicates. Asterisk indicates difference relative to Col-0 for each form at * p < 0.05.
Figure 2. Generation of QPRT RNAi lines and determination of pyridine nucleotides contents in Arabidopsis. (A) The RNAi construct pHY6 contained a 35S promoter, a sense fragment of QPRT cDNA from 164 to 506, the PDK intron, the QPRT fragment in antisense orientation, and an OCS terminator. (B) QPRT mRNA levels analyzed by qRT-PCR. Contents of NAD(H) (C) and NADP(H) (D) were quantified. White bars, reduced forms; black bars, oxidized forms. Data are means ± SE of three biological replicates. Asterisk indicates difference relative to Col-0 for each form at * p < 0.05.
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Figure 3. Reduced transcript levels of QPRT are associated with increased susceptibility. (A) The QPRT RNAi plants showed enhanced sensitivity to Pst-avrRpt2 compared with Col-0. Leaves from two 5-week-old independent lines of QPRT RNAi or Col-0 were inoculated with Pst-avrRpt2 at a concentration of 5 × 105 cfu·mL−1. Bacterial growth was measured at 0, 24, and 48 hpi. 1, Col-0; 2, At-pHY6#2; 3, At-pHY6#13. Expression of ICS1 (B), PR1 (C) and PR5 (D) was measured by qRT-PCR in Arabidopsis challenged with Pst-avrRpt2 at 48 hpi. (E) Quantification of SA in pathogen-challenged plants at 48 hpi. Means ± SE of three repetitions are shown. Asterisk indicates means are significantly different from those of Col-0 at p < 0.05.
Figure 3. Reduced transcript levels of QPRT are associated with increased susceptibility. (A) The QPRT RNAi plants showed enhanced sensitivity to Pst-avrRpt2 compared with Col-0. Leaves from two 5-week-old independent lines of QPRT RNAi or Col-0 were inoculated with Pst-avrRpt2 at a concentration of 5 × 105 cfu·mL−1. Bacterial growth was measured at 0, 24, and 48 hpi. 1, Col-0; 2, At-pHY6#2; 3, At-pHY6#13. Expression of ICS1 (B), PR1 (C) and PR5 (D) was measured by qRT-PCR in Arabidopsis challenged with Pst-avrRpt2 at 48 hpi. (E) Quantification of SA in pathogen-challenged plants at 48 hpi. Means ± SE of three repetitions are shown. Asterisk indicates means are significantly different from those of Col-0 at p < 0.05.
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Figure 4. Contents of pyridine nucleotides in Col-0 and RNAi plants upon Pst-avrRpt2 infection. Levels of NAD (A), NADH (B), NADP (C) and NADPH (D) in bacterium-challenged plants at 48 hpi were determined. Data are means ± SE of four independent extracts. Asterisk indicates significant differences from Col-0. * p < 0.05.
Figure 4. Contents of pyridine nucleotides in Col-0 and RNAi plants upon Pst-avrRpt2 infection. Levels of NAD (A), NADH (B), NADP (C) and NADPH (D) in bacterium-challenged plants at 48 hpi were determined. Data are means ± SE of four independent extracts. Asterisk indicates significant differences from Col-0. * p < 0.05.
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Figure 5. Transcript abundance of oxidative stress marker genes were suppressed in the RNAi plants compared with Col-0 at 48 hpi. Transcriptional expression is shown relative to the reference gene ACTIN2. Values shown indicate means of four biological replicates. Asterisks indicate that values are significantly different from those of Col-0. * p < 0.05.
Figure 5. Transcript abundance of oxidative stress marker genes were suppressed in the RNAi plants compared with Col-0 at 48 hpi. Transcriptional expression is shown relative to the reference gene ACTIN2. Values shown indicate means of four biological replicates. Asterisks indicate that values are significantly different from those of Col-0. * p < 0.05.
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Li, S.; Ding, H.; Deng, Y.; Zhang, J. Knockdown of Quinolinate Phosphoribosyltransferase Results in Decreased Salicylic Acid-Mediated Pathogen Resistance in Arabidopsis thaliana. Int. J. Mol. Sci. 2021, 22, 8484. https://doi.org/10.3390/ijms22168484

AMA Style

Li S, Ding H, Deng Y, Zhang J. Knockdown of Quinolinate Phosphoribosyltransferase Results in Decreased Salicylic Acid-Mediated Pathogen Resistance in Arabidopsis thaliana. International Journal of Molecular Sciences. 2021; 22(16):8484. https://doi.org/10.3390/ijms22168484

Chicago/Turabian Style

Li, Shengchun, Haiyan Ding, Yi Deng, and Jiang Zhang. 2021. "Knockdown of Quinolinate Phosphoribosyltransferase Results in Decreased Salicylic Acid-Mediated Pathogen Resistance in Arabidopsis thaliana" International Journal of Molecular Sciences 22, no. 16: 8484. https://doi.org/10.3390/ijms22168484

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