Identification of nitric oxide mediated defense signaling and its microRNA mediated regulation during Phytophthora capsici infection in black pepper

Abstract

Nitric oxide plays a significant role in the defense signaling during pathogen interaction in plants. Quick wilt disease is a devastating disease of black pepper, and leads to sudden mortality of pepper vines in plantations. In this study, the role of nitric oxide was studied during Phytophthora capsici infection in black pepper variety Panniyur-1. Nitric oxide was detected from the different histological sections of P. capsici infected leaves. Furthermore, the genome-wide transcriptome analysis characterized typical domain architect and structural features of nitrate reductase (NR) and nitric oxide associated 1 (NOA1) gene that are involved in nitric oxide biosynthesis in black pepper. Despite the upregulation of nitrate reductase (Pn1_NR), a reduced expression of Pn1_NOA1 was detected in the P. capsici infected black pepper leaf. Subsequent sRNAome-assisted in silico analysis revealed possible microRNA mediated regulation of Pn1_NOA mRNAs. Furthermore, sRNA/miRNA mediated cleavage on Pn1_NOA1 mRNA was validated through modified 5' RLM RACE experiments. Several hormone-responsive cis-regulatory elements involved in stress response was detected from the promoter regions of Pn_NOA1, Pn_NR1 and Pn_NR2 genes. Our results revealed the role of nitric oxide during stress response of P. capsici infection in black pepper, and key genes involved in nitric oxide biosynthesis and their post-transcriptional regulatory mechanisms.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-024-01414-z.

Introduction

Nitric oxide (NO) is a key signaling molecule having profound effect on immune responses of plants and animals (Besson-Bard et al. 2008; Wendehenne et al. 2001). Nitric oxide burst is one of the first layer responses observed during pathogen infection. The early induction of nitric oxide activates critical immune responses in plants against pathogens (Sun et al. 2021), by modulating the chemical interactions between locally controlled accumulation of Reactive Nitrogen Species (RNS) and proteins participating in the signaling networks of defense responses (Bellin et al. 2013). The virulence factor cryptogein produced during the infection of Phytophthora cryptogea was detected as the effective initiators of nitric oxide and subsequent host cell death (Lamotte et al. 2004). Furthermore, nitric oxide is found to modulate the defense response of potato leaves challenged with avirulent race of P. infestans (Abramowski et al. 2015). The role of nitric oxide was also established in response to hyper sensitive cell death against bacterial pathogen (Delledonne et al. 2001; Mur et al. 2008) and necrotrophic fungal pathogens (Perchepied et al. 2010; Yoshioka et al. 2009). Nitric oxide also plays a multi-dimensional role in drought tolerance by enhancing antioxidant systems, restricting water loss through ABA-mediated stomatal response and regulating the expression of drought responsive genes (Khan et al. 2019; Castillo et al. 2018). The basic mode of action of nitric oxide is through complex networks of second messengers such as Ca²⁺, cyclic GMP (Guanosine 5'-Monophosphate), cyclic ADP (Adenosine 5'-diphosphate)-ribose and lipids (Jeandroz et al. 2016). The intracellular level of nitric oxide is largely dependent on the balance between its synthesis and metabolism. The chemical interactions of nitric oxide to the specific residues on the target protein further undergoes nitric oxide-dependent post-translational modifications (Besson-Bard et al. 2008). Among these post-translational modifications, S-nitrosation is important in transducing nitric oxide bioactivity during stress response and development process (Sun et al. 2021; León and Costa-Broseta 2020; Lubega et al. 2021; Yu et al. 2014).

Nitric oxide is synthesized by the enzyme nitric oxide synthase (NOS) in L-Arginine pathway in animals. Despite a homolog of animal NOS identified in unicellular green algae Ostreococcus tauri (Foresi et al. 2010), the higher plants do not possess a gene or protein with sequence similarity to animal or bacterial NOS (Jeandroz et al. 2016). Even though a NOS-like enzyme (AtNOS1) was identified in Arabidopsis thaliana, it was renamed as Nitric oxide associated protein 1 (AtNOA1) as it was unable to bind and oxidize arginine to nitric oxide and confirmed as a member of the circularly permuted GTPase family (cGTPase). Nitric oxide can also be generated by the enzyme nitrate reductase that is involved in the nitrate assimilation. Nitrate Reductase (NR; EC 1.6.6.1) is a multi-center electron transfer protein catalyzing NAD(P)H-dependent reduction of nitrate to nitrite. The NR-mediated nitric oxide production has been reported in stomatal closure (Bright et al. 2006; Desikan et al. 2002), cold acclimation and freezing tolerance in Arabidopsis (Zhao et al. 2007), pathogen infection in plants (Caamal-Chan et al. 2011; Yamamoto-Katou et al. 2006).

MicroRNAs fine-tune the immune responses in plants upon recognition of PAMPs (Pathogen Associated Molecular Patterns) and pathogen effectors (Katiyar-Agarwal & Jin 2010; Ruiz-Ferrer & Voinnet 2009). MicroRNAs regulate the expression of disease resistance related genes and modulate pathways of hormone signalling and production of reactive oxygen species (Muhammad et al. 2019; Zhang et al. 2023). The role of miRNAs in biotic stress response was first characterized from Arabidopsis thaliana during the interaction of bacterial pathogen Pseudomonas syringae pv. tomato (Pst) (Navarro et al. 2006). Subsequent studies revealed the modulation of miR393 on salicylic acid (SA) auxin balance (Robert-Seilaniantz et al. 2011) which in turn re‐directs secondary metabolic flow and the exocytosis of PR1 (pathogenesis-related protein 1) (Zhang et al. 2011) to counteract pathogen infection in plants. Many studies revealed the critical role of microRNAs during disease pathogenesis by fungal invasion in plants (Yin et al. 2012; Wong et al. 2014; Chen and Cao 2015). Furthermore, the microRNA mediated defence regulation was reported during Phytophthora infection in tomato (Luan et al. 2015, 2016) and soybean (Wong et al. 2014). The alteration of microRNA expression pattern was reported on exogenous application of NO in Alfalfa plants (Zhao et al. 2020). NO mediated miRNAs are involved in signalling and cross-talk with biotic and abiotic stress regulators (Zhou et al. 2021).

Quick wilt disease caused by P. capsici is a major threat affecting black pepper cultivation and leads to severe crop loss (Asha and Soniya 2016). Panniyur-1 is one of the farmers-preferred, high yielding variety of black pepper, widely cultivated in the plantations of India. The susceptibility of Panniyur-1 plants to quick wilt disease cause extensive plant mortality and hence low productivity. The high throughput leaf sRNA transcriptome analysis revealed fifty conserved and four novel microRNAs from black pepper (Asha et al. 2016). Along with this, the critical role of small RNAs derived from non-coding structural RNAs such as transfer RNAs (Asha and Soniya 2016) and ribosomal RNAs (Asha and Soniya 2017) were also elucidated during Phytophthora infection in black pepper. In the present study, we analyzed nitric oxide production in black pepper variety Panniyur-1 in response to the pathogen signals from Phytophthora capsici and a transcriptome-assisted analysis was carried out for the characterization of genes involved in nitric oxide synthesis. The key genes involved in nitric oxide synthesis such as nitric oxide associated protein (NOA1) and nitrate reductase (NR) were analyzed for their expression pattern upon pathogen infection. Furthermore, the high throughput sRNAome of P. capsici infected and control uninfected samples were analysed to identify the possible miRNA/small RNA mediated targeting of key genes involved in nitric oxide biosynthesis pathway in black pepper.

Materials and methods

Plant material and stress treatment

Piper nigrum variety Panniyur-1 was maintained in the green house at Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram. The virulent cultures of P. capsici was obtained from the Department of plant pathology, College of Agriculture, Vellayani, Thiruvananthapuram and the culture were maintained in the potato dextrose medium on continuous sub-culturing. Plants were infected with 48 h old, actively growing mycelium discs (5 mm diameter) on the adaxial side of leaves and on the collar region of the plants (Asha and Soniya 2016). The control plants were inoculated with the PDA medium without any mycelium. The nitric oxide content was analyzed from the infected leaf and stem tissues at 24 h post infection (24hpi).

Detection of nitric oxide and nitrate reductase activity during P. capsici infection

The detection of nitric oxide from the plant tissues was done as previously reported (Requena et al. 2005). In brief, thin sections were prepared from the infected and control plant parts and washed in 1 mL of Tris–HCL buffer (10 Mm, pH 7.2). The sections were then incubated in 1 ml of 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate (DAF-FM DA) solution (10 µM) at room temperature and darkness for 1 h. After incubation, the sections were washed three times and mounted on microscopic slides. In order to verify nitric oxide specificity of DAF-FM, the infected stem sections were incubated in nitric oxide scavenger solution (diethyl thio carbamate solution (100 µM) (Harbrecht 2006; Singh and Bhatla 2017) for 1 h and stained with 10 µM DAF-FM DA in 10 mM Tris–HCL (pH 7.2). The different zones of sections (epidermis, cortex and vascular bundles) were analyzed for nitric oxide production using confocal laser scanning microscope (NIKON A1R-A1) with filter set at excitation 450–490; emission at 500–550 and images were acquired.

The nitrate reductase assay (Harris et al. 2000) was performed from the black pepper leaves at 0,12, and 24 h after P. capsici infection. Leaf tissues (0.2 g) were ground into a fine powder with a mortar and pestle using liquid nitrogen and immediately suspended in 1 mL of extraction buffer (Tris HCl 50 mM pH 8.2, EDTA 5 mM, cysteine 5 mM,1% PVP (w/v), and 2 mM of β-mercaptoethanol). The crude extract was centrifuged at 14,000g, 4 °C for 15 min, and the clear supernatant was used for the protein estimation and the measurement of enzyme activity. After mixing the supernatant with reaction buffer (50 mM Na₂HPO₄ (pH 7.5), 10 mM KNO₃, and 0.5 mM NADH) and incubating for 10 min, the reaction was stopped by adding 1 ml of 1% sulphanilic acid in 2 M HCl (v/v) and 1 ml of 0.02%N-(1-Naphthyl) ethylene diamine dihydrochloride. The optical density at 540 nm was read after 15 min. The amount of nitrite formed was calculated from a standard curve plotted using the A540 values obtained from the known amounts of nitrite. While the protein concentration was estimated according to Bradford’s method (Bradford 1976) using BSA as standard.

Genome wide analysis of key genes involved in nitric oxide biosynthesis from black pepper

The nucleotide and protein sequences of Arabidopsis NOA1 (AT3G47450), NIA1/NR1 (AT1G77760) and NIA2/NR2 (AT1G37130) genes (Vazquez et al. 2019; Xie et al. 2013; Zhao et al. 2009) were retrieved from TAIR database (Berardini et al. 2015) and used as a query against the black pepper genome (Hu et al. 2019) to identify the potential homologs using BLASTN/BLASTP. The full-length coding genes of NOA, NIA1/NR1, NIA2/NR2 with E-value cut off 1e−05 were extracted from the genomic scaffolds and the exon–intron regions were annotated. The coding sequences were translated and the molecular weight and pI of PnPR-1 proteins were estimated by the ExPASy ProtParam tool (https://web.expasy.org/protparam). Functional domains in the deduced full-length protein sequences were identified using NCBI CDD, SMART and TREND web resources (Marchler-Bauer et al. 2005; Letunic et al. 2021; Gumerov and Zhulin 2020). The gene ontology (GO) and KEGG pathway analysis was subsequently carried out using PANNZER2 and BlastKOALA web servers (Törönen et al. 2018; Kanehisa et al. 2016).

Transcriptome analysis of Panniyur-1 NOA1 (Pn1_ NOA1) and nitrate reductase (Pn1_NR1 and Pn1_NR2)

The rooted cuttings of black pepper variety Panniyur-1 was collected from College of Agriculture, Vellayani, Thiruvananthapuram and maintained in the green house of Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram. The virulent culture of P. capsici obtained from Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram was used for inducing infection, as previously reported (Asha and Soniya 2016). Total RNA was isolated from the leaf of P. capsici infected panniyur-1 plants at 24 h post infection (24hpi). The uninfected healthy leaf sample was used as the control. RNA samples with high purity (OD260/280 between 1.8 and 2.2) and RNA integrity number, RIN > 7.5 was used for mRNA library construction and Illumina HiSeq™ 2000 sequencing (BGI, Shenzhen, China). The raw reads were processed and transcriptome sequences were assembled from control leaf and P. capsici infected leaf using Trinity (Grabherr et al. 2011). The differential expression pattern of Pn1_NOA, Pn1_NR1, Pn1_NR2 genes were analyzed based on the FPKM (Fragments Per kb per Million fragments) values (Mortazavi et al. 2008) of the transcripts from control leaf and P. capsici infected leaf transcriptome. Fold change was selected based on the p-value (p > 0.005) and FDR values (FDR ≤ 0.001). The transcripts with significant hits to the reference genes (Pn1 NOA1, Pn1 NR1 and Pn NR2) were analyzed further. EMBOSS NEEDLE (a pair-wise alignment program), on the EMBL-EBI web server was used to evaluate the sequence identity (Needleman and Wunsch 1970; Chojnacki et al. 2017).

Structure and phylogenetic analysis of Pn1_NOA and Pn1_NR

To determine the structural features, homology-based protein modeling was carried out in Phyre2 (Kelley et al. 2015) and SWISSMODEL (Waterhouse et al. 2018). Nitrate reductase from Pichia angusta (PDB ID:2BIH, 48% identity), and Zea mays (PDB ID:1CNF, 68.18% identity) were selected as templates (based on Blastp against PDB with an E-value cutoff 1e−05) for modelling the Moco and FAD modules of Pn1 NR. Cyt-b5 or heme domain is modelled based on the 1.5 Å structure of House Fly Cytochrome B5 (PDB ID: 2IBJ, 38.75% identity). For modelling Pn1_NOA1, YqeH GTPase (PDB ID:3EC1, 34% identity) from Geobacillus stearothermophilus (an AtNOA1 ortholog) (Sudhamsu et al. 2008) was used. Modelled structures were refined in the ModRefiner (Xu and Zhang 2011) and the stereo-chemical quality of the structures were validated using PROCHECK module in SAVES v6.0 (https://saves.mbi.ucla.edu/) and ProSA-web (Wiederstein and Sippl 2007). PyMOL with academic license (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC) was used to visualize the structures. To analyse the charge distribution, APBS electrostatics were also calculated in PyMOL. NCBI Blast was used to find possible homologs of NOA1 and NR (E-value cut off 1e−05; query sequences Pn1_NOA1 and Pn1_NRs). Once potential homologs were validated (by reciprocal blast and SMART domain analysis), 25 NOA homologs and 33 NR homologs from different species (including lower plant taxa) were included for phylogenetic tree construction. Multiple sequence alignment (MSA) was performed in MAFFT (Katoh et al. 2019) and phylogenetic analysis was done using MrBayes 3.2.7_0 (Huelsenbeck and Ronquist 2001) in the NGPhylogeny.fr webserver (Lemoine et al. 2019). In order to display the sequence alignment and phylogenetic tree, ESPript 3.0 (Robert and Gouet 2014) as well as iTOL (Letunik et al. 2021) were used.

Real time qRT-PCR expression analysis of Pn1_NOA1 and Pn1_NR genes

The expression variation of Nitric oxide Associated protein1 (NOA1) and Nitrate reductase (NR) transcripts was studied in black pepper plants after P. capsici infection. Three biological replicates were used per treatment. Total RNA from leaf tissues of pathogen infected as well as mock inoculated control black pepper plants were isolated by the Trizol reagent (Invitrogen). cDNAs were synthesized from 1 µg of total RNA using high-capacity cDNA reverse transcription kit (Applied Biosystems). Real-time quantitative reverse-transcription (qRT-PCR) was performed using Applied Biosystems7900 HT sequence detection system using POWER SYBR Green qPCR Master Mix (ABI) according to manufactures instructions. Each qPCR reaction was conducted in 10µL volumes containing 1µL of cDNA (5 ng), 5µL of SYBR green and 5 pmol of forward and reverse primers (Supplementary Table 1) with the following conditions: 40 cycles at 95 °C for 15S and 65 °C for 15S. Negative PCR controls (No cDNA template) were prepared to detect possible contamination. The expression of these genes was compared with that of endogenous control gene (Actin). Relative mRNA ratios were calculated by 2^^ΔΔCT.

Identification of microRNA mediated regulation of Pn_NOA1 in black pepper

Panniyur-1 plants were inoculated with mycelia culture disc of the pathogen as described previously (Asha and Soniya 2016). Leaves were taken from the pathogen infected plants at 24 hpi and small RNA (≤ 200 nt) was isolated using miRVana miRNA isolation kit (Ambion, Austin, TX) according to manufacturer’s instructions. RNA samples with high purity (OD260/280 between 1.8 and 2.2) and RNA integrity number, RIN > 7.5 was used for Illumina high-throughput sequencing (BGI, Shenzhen, China). The raw reads were processed, and the clean reads within the size range of 18–30 nt were used for the analysis. The plant small RNA target server (plantgrn.noble.org/psRNATarget) was used for the identification of potential miRNA target sites on Pn1_NOA1 mRNAs. The possible miRNA mediated cleavages were validated experimentally by 5'RLM RACE experiments (Asha et al. 2016; Asha and Soniya 2017). Furthermore, potential miRNA-like hairpin precursors were predicted for the sRNAs/miRNAs from the RNA folding program of the UEA small RNA workbench (Stocks et al. 2012). The small RNA sequence data is submitted to NCBI GEO database with the accession number GSM1606153 and GSM1606154.

Analysis of cis-acting regulatory DNA elements of NOA1 and NR genes in P. nigrum

The promoter sequences (2.0kbp upstream of translation start site) of NOA1 and NR1/NR2 genes were retrieved from black pepper genome. The cis-elements were predicted from the New PLACE (Higo et al. 1999) and PlantCare (Lescot et al. 2002). The cis-elements obtained were compared with each other.

Results

Detection of nitric oxide in black pepper plants on infection with P. capsici

Nitric oxide accumulation was analyzed from thin leaf sections of the control and P. capsici infected (24hpi) plants after treatment with DAF-FM DA. The green fluorescence of the intracellular nitric oxide was detected in the epidermis, cortex and vascular bundles of the infected leaf. Furthermore, the hypersensitive regions in the infected leaf showed an increased fluorescence surrounding the vascular bundles and cortex tissues (Fig. 1a–h). Meanwhile a continuous increase in nitrate reductase activity was also observed from the leaves of P. capsici infected plants at early hours of infection (24hpi). The increase in nitrate reductase activity indicates nitric oxide production during pathogen interactions (Fig. 1i). The systemic nodal stem also showed a higher fluorescence compared to the control stem sections (Fig. 2). The fluorescence was detected high in the vascular bundles. Furthermore, the complete suppression of fluorescence were also observed in the nitric oxide scavenger treated, infected stem sections.

Fig. 1.

NO detected by confocal laser scanning microscopy shown as fluorescence in the leaf and stem sections of black pepper plants infected with P. capsici: The bright field images (a and b) and confocal microscopy images (c and d) on cross Sections from leaf lamina, midrib on DAF treated control leaf. The bright field images (e and f) and confocal microscopy images (c and d) on cross Sections from leaf lamina, midrib on DAF treated and infected leaf (24hpi) (g and h). i Nitrate reductase assay on P. capsici infected black pepper leaf. Mean Standard Deviation is plotted as error bar

Fig. 2.

Zones of medulla and vascular bundles on DAF-FM DA treated control stem (a–d: stem sections of the replicates) and (e–h) infected stem (24hpi) and NO scavenger treated infected stem section (i–l). Bright field image corresponding to the sections showing fluorescence (left) were also shown. Red arrow indicates vascular bundles

Genome-wide analysis of key genes involved in nitric oxide biosynthesis

The genome-wide analysis revealed single gene loci for Pn_NOA1, mapped to the scaffold Pn12. The gene structure analysis revealed thirteen exons in Pn_NOA1 (Fig. 3a). Meanwhile, two homologs of nitrate reductase genes (PnNR1 and PnNR2) were identified from the P. nigrum scaffold 10 and 11 respectively, with 5 and 6 exons (Fig. 3b, c). The structural and functional properties of the P. nigrum NOA1 and NR genes were listed in Table 1. The deduced P. nigrum Panniyur-1 NOA1 protein (Pn1_NOA1gene product) is a 539-amino acid residue protein with a molecular weight of 58.55KDa and theoretical pI of 9.1. GO Analysis revealed the GTP binding (GO:0005525) functions of the Pn1_NOA1 proteins. The KEGG analysis showed that Pn1_NOA1 belongs to the amino acid metabolism pathway, including Arginine biosynthesis (ko00220), Arginine and proline metabolism (ko00330), Metabolic pathways (ko01100), Biosynthesis of secondary metabolites (ko01100) and Plant-pathogen interaction (ko04626).

Fig. 3.

a–c Gene structure details of Pn_NOA, Pn_NR1 and Pn_NR2 genes. d and e Differential expression of Pn1_NOA and Pn1_NR gene transcripts during P. capsici infection in black pepper leaf

Table 1.

Structural and functional properties of Piper nigrum NOA and NR genes

Gene ID	From the reference genome of the cultivar ‘Lampung Daun Kecil'						From P. nigrum Panniyur-1 transcriptome
	Mapped genome scaffold	Length of coding genes (CDS)	Deduced protein length	MW	Number of exons	Theoretical pI	Gene ID	Assembled transcript length	Deduced protein length	MW	Theoretical pI
PnNOA1	Pn12	1620	539	58.49	13	9	Pn1_NOA1	1620	539	58.55	9.1
PnNR1	Pn10	2673	890	99.65	5	6.28	Pn1_NR1	2694	897	99.87	6.19
PnNR2	Pn 11	2625	874	97.18	6	6.14	Pn1_NR2	2697	898	100	6.14

The deduced proteins of Panniyur-1 nitrate reductases, Pn1_NR1 (99.87KDa) and Pn1_NR2 (100KDa) shared 92.2% sequence identity (Supplementary Fig. 1). In terms of GO analysis, three biological processes, six molecular functions and two cellular components were identified for nitrate reductase proteins. Among the biological processes, Pn1_NR have functional role in oxidation–reduction process (GO:0055114), nitric oxide biosynthetic process (GO:0006809) and nitrate assimilation (GO:0042128). Meanwhile nitrate reductase (NADPH) activity (GO:0050464), FAD binding (GO:0071949), molybdenum ion binding (G0:030151), molybdopterin cofactor binding (GO:0043546), nitrate reductase (NADH) activity (GO:000970) and heme binding (GO:0020037) activities were detected in the term molecular function. Based on KEGG annotation analysis, Pn1_NR1 and Pn1_NR2 belong to the energy metabolism pathway (K10534) that includes Nitrogen metabolism (ko00910), metabolic pathways (ko01100) and microbial metabolism in diverse environments (ko01120).

Transcriptome analysis of NOA and NR genes from Panniyur-1

The differential expression of Pn1_NR and Pn1_NOA1 genes were analyzed from the leaf transcriptomes of control and P. capsici infected Panniyur-1 plants (Supplementary Table 2). P. capsici infected leaf (Pn_IL) showed decrease in expression of Pn1_NOA1 transcripts (Unigene10512 and Unigene5828) compared to the control (Fig. 3d), while all the Pn1_NR transcripts (CL10507.Contig2, CL7569.Contig1, CL7569.Contig2, CL7569.Contig3) were upregulated in infected leaf (Fig. 3e). Among these, a significant up-regulation was detected for CL10507.Contig2.

Structure and phylogenetic analysis of Pn1_NOA and Pn1_NR

The modelled structures of Pn1_NOA1 and Pn1_NR (Fig. 3c–e, 4b–g) are of high quality and reliable (overall quality factor > 90, and more than 95% residues in the models are in the allowed region, upon model evaluation). The Pn1_NOA1 structure adopted similar structural fold as that of GTPase_ YqeH with three conserved structural domains namely i) Zn binding domain (ZBD), ii) circularly permuted G-motif (CPG) domain with G1 to G5 motifs (guanine nucleotide-binding regions), and iii) C-terminal domain (CTD) (Fig. 3a–e). ZBD containing four conserved cysteine (C) residues (Liu et al. 2010) which are absolutely preserved in Pn1_NOA1 too (Fig. 4a, b). As this N-terminal ZBD region is highly disordered, structural template is not available yet to model the ZBD area. CPG domain (region 165 to 342) is a typical G-protein fold composed of centrally located β-strands, surrounded by α-helices. Conserved guanine nucleotide binding motifs (G1-G5) are positioned within this CPG domain (Fig. 4c), which has significant roles in nucleotide exchange and conformational change (Bourne et al. 1991; Sudhamsu et al. 2008). Positive charge residues are accumulated in the GDP binding cleft of the CPG domain surface and these residues play significant role in binding activities (Fig. 4e). Pn1 NOA1 also showed certain sequence insertion as previously reported in A. thaliana (Moreau et al. 2008), however as they are positioned in loop/turn areas it does not affect the overall folding pattern (Supplementary Fig. 2). Furthermore, a signal peptide was predicted in the N-terminal region of Pn1_NOA (cleavage occurs in 31 to 32 amino acid areas), and suggested as a cytoplasmic protein (PSORT score 0.480).

Fig. 4.

Sequence/structural features of Pn1_NOA1 a domain arrangement in Pn1_NOA1. b Alignment of Pn1_NOA protein with Arabidopsis NOA1 and the Geobacillus stearothermophilus YqeH GTPase (an AtNOA1 ortholog, used as structural template). The conserved domains (ZBD, CPG and CTD) are highlighted. Conserved cysteine (C) residues in ZBD are marked with asterisks. c CPG and CTD domains of Pn1_NOA1 (Homology model based on GsYqeH) d surface view of GsYqeH with GDP in the binding cleft. e Surface view of Pn1_NOA1 with the positively charged binding cleft (marked with arrow)

Pn1_NR1 and Pn1_NR2 are homodimeric (subunit size ~ 100KDa) cytosolic proteins (PSORT score 0.450) with the structural conformation comprises specific domains such as i) Oxidoreductase molybdopterin binding domain (PF00174), ii) Mo-co oxidoreductase dimerisation domain (PF03404), iii) Cytochrome b5-like Heme/Steroid binding domain (PF00173) iv) Oxidoreductase NAD-binding domain (PF00175) and v) Oxidoreductase FAD-binding domain (PF00970) (Fig. 5a). Three major cofactors of NRs such as Molybdopterin, iron-heme and FAD are found, respectively, in; Moco domain (first two domains of NR (i and ii) together), heme binding Cytochrome b5 domain and Cytochrome b reducing fragment (last two domains—iv and v).The full-length NR structure was not yet available, therefore, N-terminal Moco-domain, middle Cyt-b6/heme binding domain and C-terminal cytochrome b5 reductase domain were specifically modelled based on the available structural templates (Fig. 5b–g). Structural model of Pn1_NR1 N-terminal nitrate reducing module (NR-Mo or Moco-domain) based on homology adopted the similar structural fold of NR-Mo of P. angusta (Ogataea angusta) assimilatory nitrate reductase (Fischer et al. 2005). The C terminal module of Pn1_NR1 (637 to 898) shows identical structural conformation of corn (Z. mays) NR-cytochrome b5 reductase fragment (NR-CbR) (Lu et al. 1995). Like corn NR-CbR, C-terminal fragment of Pn1_NR1 also consist of two specific domains, where its N-terminal area is consists of six β-strands (anti-parallel), which adopts a Greek key motif conformation. C-terminal area adopts a Rossmann fold (alternating motif of β/α/β strand) like topology, which is a typical characteristic feature of FAD and NADH binding proteins (Hanukoglu 2015). The middle Cyt-b6/heme binding domain of Pn1_NR1 possess similar structural fold of House fly (Musca domestica) cytochrome b5, with substantial negative charge accumulation in the heme binding area. Similarly, Pn1_NR2 proteins also possess the identical structural features (Fig. 5g).

Fig. 5.

Domain organization and structural features of P. nigrum NR a domain arrangement in Pn1_NR and its homologs. b Homology model of Pn_NR1 Moco domain (homodimer). c Electrostatic charge distribution in the Pn_NR1 Moco domain surface. Arrow indicates the binding cleft (positive charge). d and e Cartoon and surface representation (with charge) of Cyt-b5 domain of Pn_NR1. Here the heme binding cavity is negative in charge. f and g Cartoon and surface representation (with charge) of Cytochrome b reducing fragment of Pn_NR1

Pn1_NOA1 and its potential homologs from selected Viridiplantae members (including basal angiosperms and lower plant taxa) displayed distinct grouping pattern and Pn1_NOA1 is clustered in between the basal Magnoliophyte (Ambroella) and liliopsida (Fig. 6a) when the homolog from the unicellular algae Chlamydomonas used as the outgroup. This branching correlates with the "paleoherb hypothesis" of magnolides, where the basal angiosperm plants display a mix of monocot and dicot feature. Pn1_NRs (Fig. 6b) constituted a separate group between eudicotyledons and liliopsida (monocotyledons), which also confirmed its mixed features and position on the evolutionary lines.

Fig. 6.

Phylograms for a Pn1_NOA1 and b Pn1_NRs with their homologs. Phylogenetic tree constructed based on Bayesian inference on MrBayes in the NGPhylogeny.fr webserver

Experimental validation of Pn1_NOA1 and Pn1_NR genes and their microRNA mediated regulation

Pn1_NR genes were found upregulated in P. capsici infected black pepper leaf compared to the control (Fig. 7). Meanwhile, the expression of Pn1_NOA1 was downregulated in the infected plant. The infected root also showed similar expression pattern as seen in the infected leaf (Supplementary Fig. 3). Moreover, the qRT-PCR showed consistent results as that of the transcriptome analysis. The raw data of qPCR analysis is provided as Supplementary Table 3. The reduced expression of Pn1_NOA1 further indicates the possibility of miRNA mediated post-transcriptional regulation in the infected plants. Subsequent analysis of the high throughput small RNA libraries of black pepper revealed a set of potential small RNA candidates that can target Pn1_NOA1 mRNAs (Supplementary Table 4). Furthermore, miRNA-like hairpin precursors were predicted for certain sRNA candidates (Supplementary Table 5 and Supplementary Fig. 4). Modified 5'RLM RACE experiment could detect miRNA mediated cleavage on Pn1_NOA1 mRNAs (Fig. 8a). Cleavage were mapped at the Zn binding domain and CTD domain. The red arrow indicates the cleavage sites mapped from Pn1_NOA1 mRNAs from the RACE experiments. The sequence conservation of miRNA binding sites of Pn1_NOA1 to its counterparts from other plant lineages revealed distinct sequence variation (Fig. 8b), indicating the possibility of black pepper specific regulatory mechanism.

Fig. 7.

Quantitative real-time PCR analysis of Pn1_NOA1 and Pn1_NR genes upon P. capsici infection (24hpi) in black pepper plants. The error bars represent SD (n = 3)

Fig. 8.

a miRNA mediated cleavage detected from the Pn1_NOA1. The red arrow indicates the cleavage sites detected at CTD from the modified 5'RLM RACE experiments. b The sequence conservation of miRNA binding sites of Pn1_NOA1 to other plant species

Identification of cis-regulatory elements of Pn_NOA1 and Pn_NR genes

Apart from the conservative core promoter elements such as CAAT box and TATA Box, different hormone-responsive cis-regulatory elements were predicted through in silico analysis of promoter regions of Pn_NOA1 and Pn_NR genes (Supplementary Table 6). AuxRR-core, the cis-regulatory element (GGTCCAT) involved in auxin-response and Myb-binding site involved in drought response (CAACTG) was found in Pn_NR1, while two cis-regulatory elements involved in the MeJA response (TGACG-motif and), salicylic acid (TCA-elements) and gibberellin response (P-box) were detected in Pn_NR2 (Fig. 9). Similarly, the cis-acting element ABRE, responsible to abscisic acid, TCA-element for the salicylic acid, TATC-box for gibberellin response were found in Pn_NOA1 gene promoters. The results from the plantCare database revealed that Pn_NOA1 and Pn_NR genes possess many stress inducible Myb-binding sites in the promoter regions. Other cis-elements essential for meristem induction (CAT box/NON box), light response (Sp1/GT1-motif/AT1-motif/Box4/TCCC-motif/G-box) and anaerobic induction (GC-motif/ARE) was commonly detected in both Pn_NR1 and Pn_NR2 genes.

Fig. 9.

Cis-regulatory elements identified from the promoter regions of Pn_NOA1, Pn_NR1 and Pn_NR2 genes

Discussion

Nitric oxide is a gaseous, secondary messenger that possess wide range of physiological functions in plants and have critical role in physiological processes such as growth, development, metabolism, leaf senescence, abiotic and biotic stress responses (Astier et al. 2018; Mur et al. 2013). Being a molecular messenger, nitric oxide modulates expression of several genes involved in hormone signaling and stress responses (Freschi 2013). In the present study, the stress responsive role of nitric oxide and its plausible mechanisms of synthesis was analyzed on P. capsici infection in black pepper plants. Consistent with previous reports (Mur et al. 2005), nitric oxide was found to be induced in the leaves of black pepper plants during early stages of infection (24hpi) by staining with DAF-FM DA. DAF-FM DA was widely used fluorophore to localize nitric oxide in plant tissues as it reacts with N₂O₃, a by-product of nitric oxide oxidation and results in highly fluorescent DAF-FM Triazole (DAF-FM-T) (Kojima and Nagano 2000). DAF-DA is a specific chromophore that can permeates the cell membrane, where it is quickly transformed into impermeant form and the fluorescent quantum yield increases once it reacts with nitric oxide (Kojima and Nagano 2000). As previously reported, the complete suppression of fluorescence in the nitric oxide scavenger treated, infected stem sections revealed DAF-FM DA as a nitric oxide specific fluorophore (Planchet et al. 2014). The early production of nitric oxide contributes to disease resistance in plants during P. capsici infection (Caamal-Chan et al. 2011; Requena et al. 2005). Mostly, nitric oxide is produced as a transient burst during pathogen and elicitor responses in plants, and its continuous synthesis in later stages triggers a sequence of events leading to defense response (Planchet and Kaiser 2006). Besides the signal function, nitric oxide act as toxic molecule in the plant cell. The unregulated nitric oxide production causes cell death through oxidative stress, disrupted energy metabolism, DNA damage, activation of poly (ADP-ribose) polymerase or dysregulation of cytosolic Ca²⁺ (Delledonne et al. 2001).

The two routes of nitric oxide production have different sensitivity to biotic and abiotic stresses. The reductive route of nitric oxide synthesis is mediated by nitrate reductase (NR) enzymes, while nitric oxide synthase and nitric oxide associated protein1 (NOA1) was reported to mediate the oxidative route of nitric oxide synthesis in plants (Astier et al. 2018; Besson-Bard et al. 2008). Furthermore, cytosolic nitrate reductase (NR) was found as a major source of nitric oxide during plant–pathogen interactions (Modolo et al. 2005; Mur et al. 2012). As previously reported from Arabidopsis (Xie et al. 2013), genome-wide analysis of black pepper revealed two homologs of nitrate reductase genes such as Pn_NR1 and Pn_NR2. Nitrate reductase is a necessary partner for nitric oxide production, and the multi-domains that act as redox centers (Campbell 1999; Tejada-Jimenez et al. 2019) were detected from P. nigrum nitrate reductase genes. The extensive analysis of 1000 plant transcriptome revealed complete absence of canonical NOS in land plants (Jeandroz et al. 2016). Meanwhile, the existence of nitric oxide synthase in photosynthetic algal species reveals the possibility of NOS gene being lost during evolution of embryophytes (Astier et al. 2018; Santolini et al. 2017). AtNOA1 (nitric oxide associated 1), a functionally characterized GTPase was widely used in the study of NOS-like activity in plants as it has role on nitric oxide production (Astier et al. 2018) and indirectly linked to biotic stress response (Mwaba and Rey 2017). Meanwhile, consistent with previous reports from Arabidopsis (Moreau et al. 2008), the gene encoding P. nigrum nitric oxide associated protein-1 (Pn_NOA) was deduced to have Zinc Binding Domain (ZBD), CPG domain and CTD. The N-terminal ZBD motif belong to the treble clef family of zinc finger domains that binds nucleic acids, proteins, and small molecules during the catalysis of phosphodiester bond hydrolysis (Anand et al. 2006). These spatially close G1-G5 regions (order: G4-G5-G1-G2-G3) were found associated within the loop, and have significant roles in nucleotide exchange, GTP hydrolysis and conformational change (Sudhamsu et al. 2008).

Subsequently, the transcriptome-assisted, differential expression analysis revealed upregulation of nitrate reductase gene transcripts such as CL10507.Contig2, CL7569.Contig1, CL7569.Contig2, CL7569.Contig3 during P. capsici infection in leaf tissues of Panniyur-1 plants. The accumulation of nitrate reductase transcripts in response to early infection of P. capsici was previously reported as an incipient strategy of the host plant to fight against pathogens (Caamal-Chan et al. 2011). Furthermore, the differential expression pattern of NR genes were directly correlated with the production of nitric oxide. Meanwhile Pn1_NOA transcripts were downregulated in the leaf tissues of infected plants. During the induction of plant defense responses, nitric oxide modulates salicylic acid (SA) signaling through S-nitrosylation at cys156 of NPR1 (Non-expressor of Pathogenesis-Related Gene 1 that act as translational activator of PR-1 genes (Freschi 2013; Mur et al. 2013; Zhao et al. 2015). Down regulation of NPR1 genes during early stages of P. capsici infection (24hpi) was previously reported in black pepper variety Panniyur-1 during P. capsici infection (Asha and Soniya 2016). Besides these, nitric oxide was also reported to crosstalk with other plant hormones such as jasmonic acid (JA), polyamines, and brassinosteroids during plant defense responses (León and Costa-Broseta 2020).

Transcriptional regulation of specific genes is mediated by the interaction of transcription factors to the specific cis-regulatory elements in the genes. Different kinds of cis-regulatory elements were identified for the Pn_NOA and Pn_NR genes. Cis regulatory elements such as Myb binding sites, Myc recognition elements, TCA motif involved in salicylic acid response, Box4 involved in light response were commonly found in the promoter region of P. nigrum nitrate reductase and nitric oxide associated 1. The presence of Myb transcription factor binding sites in the promoter regions of these genes indicate their role in hormone signaling, stress responses and specialised metabolic processes such as flavonoid biosynthesis (Sheshadri et al. 2016). Meanwhile the TATC-box, TCA element and ABRE distributed in the promoter regions of Pn_NOA1 indicates its key functions in regulating the signaling pathways of salicylic acid gibberellin and abscisic acid respectively (Jiang et al. 2014). Gene expression and subsequent biosynthesis of proteins are not only dependent on the presence of cis-elements, but also on the other trans-acting modulators like microRNAs. Transcription factors and microRNAs can function as specific regulatory switches that determine the gene expression. Furthermore, during stress responses nitric acid acts as messenger between stress perception and subsequent miRNA mediated post-transcriptional gene regulation in plants. Nitric oxide interacts with reactive oxygen species and mediates downstream hormone signaling pathways (Mur et al. 2013; Singh et al. 2017). Recently exogenous nitric oxide-responsive miRNAs were detected from Alfalfa (Medicago sativa L.) under drought stress conditions (Zhao et al. 2020). P. nigrum sRNAome assisted analysis revealed potential miRNAs/sRNAs targeting Pn1_NOA mRNAs that were downregulated on pathogen infection. Subsequent in silico analysis revealed novel black pepper specific microRNAs/sRNAs potential to target Pn1_NOA mRNAs, which was further validated by the modified 5' RLM RACE experiments. Meanwhile distinct sequence diversity was observed at the miRNA binding sites of Pn1_NOA with different plant species.

In conclusion, our study revealed the role of nitric oxide during black pepper-Phytophthora interactions. The genome-wide transcriptome analysis revealed Pn_NOA1 and Pn_NR genes that are critical for the nitric oxide biosynthesis pathway in black pepper and their differential expression during pathogen infection. The nitrate reductase genes showed up-regulation in the infected leaf of the susceptible Panniyur-1 plant, meanwhile nitric oxide associated − 1 was downregulated during infection, for which miRNA mediated cleavage was validated. The functional studies of these key genes and micro RNAs can provide the exact molecular mechanism of NO biosynthesis during stress responses of black pepper.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

AS and EVS designed the work. AS, DK, MV performed the experiments and data analysis. AS wrote the paper and all authors read the paper.

Declarations

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.