Sodium chloride primes JA-independent defense against Spodoptera litura (Fabricius) larvae in Arabidopsis thaliana

ABSTRACT

Priming for better defense performance is an important strategy in acclimation to the ever-changing environment. In the present study, defense priming induced by sodium chloride at the seedling stage significantly increased the expression of defense gene VSP2, the content of total glucosinolates and the level of the reactive oxygen species in mature Arabidopsis thaliana plants after transferred into the stress-free environment. The previously primed plants could effectively resist the feeding of Spodoptera litura (Fabricius) larvae. Salt-priming enhanced defense of Arabidopsis plants in the absence of either MYC2 or AOS, which encodes a critical transcription factor in JA-signaling and an important enzyme in JA biosynthesis, respectively. Our results supported the JA-independent defense primed by sodium chloride, as well as the elevated ROS and glucosinolate level in primed plants. In addition, the feasibility of using mild salt-priming to improve crop performance in field was proposed.

KEYWORDS:

• Sodium chloride
• defense priming
• herbivory
• jasmonic acid pathway
• Arabidopsis thaliana

Introduction

Herbivory by insect larvae causes tremendous loss of crop yield and is the main threat to food supply around the world. Different strategies, such as application of pesticide, conventional breeding, and genetic engineering have been employed aiming at reducing the loss caused by insect pest in crops.¹ In pursuit of safer pest control techniques, many studies propose that priming treatments to enhance plant defense against stresses is highly promising. Compared to non-primed plants, primed plants are provided better leverage with acquired ability to resist future abiotic or biotic stresses.^2,3 The protective responses of plants in a priming state will be faster or stronger when a stress is encountered.^4–7

A growing body of literature indicates that plants primed by chemical inducers are effectively resistant to insects. In response to herbivores damaging a single leaf, the whole plant releases a bouquet of volatiles, which induce the neighboring plants into a priming state and enable plants to respond more effectively when challenged with secondary attack.⁸ The non-protein amino acid β-aminobutyric acid (BABA) is known as a potent agent to prime plants against herbivores, such as tobacco hornworms (Manduca sexta), green peach aphids (Myzus persicae), pea aphids (Acyrthosiphon pisum), and spider mites (Tetranychus urticae).^9–12 Soil or foliar application of silicon enhances resistance of plants to insect herbivores and other arthropods.¹³

Sodium chloride (NaCl), a low-cost inorganic compound, is also used as a priming agent. Priming chickpea seeds with NaCl increased the activities of amylase, invertases, sucrose synthase and sucrose phosphate synthase in shoots of primed seedlings.¹⁴ Alfalfa seeds primed with salt solution showed increased seed germinative vigor under salinity stress.¹⁵ Cakile maritima seeds imbibed in high salinity (200 mM NaCl) showed higher germination percentage and faster germination than the mock-treated seeds.¹⁶ Cakile maritima plants pre-exposed to salinity preserved a lower oxidative stress when subsequently exposed to salinity and maintain constant tolerance to salt stress.¹⁷ Arabidopsis plants exposed to repetitive salt stress were more resistant to virulent bacteria than those grown in a more stable environment.¹⁸

Upon chewing herbivory, the performance of attacked plants is orchestrated by plant hormones, which largely depends on the biosynthesis and signaling of jasmonates (JA).^19–21 Allene oxide synthase (AOS) is a key enzyme that catalyzes the formation of allene oxides from fatty acid hydroperoxides generated by lipoxygenases (LOXs), which is essential for JA synthesis.^22,23 MYC2 is a basic helix-loop-helix (bHLH) transcription factor that directly regulates expression of JA-responsive genes including VSP2.²⁴ A functional MYC2 is required for intact JA signaling and JA-mediated defense against herbivory.^25,26

Though many reports focused on NaCl-induced priming, few of them reported that priming induced by NaCl at seedling stage could impart and maintain defense “memory” against insect herbivores in grown-up plants. Moreover, the relationship between NaCl-induced defense priming and the JA-signaling pathway should be reconsidered as full-scale activation of the JA-induced defense often disrupts the balance between defense and growth. As shown in Figure S1 in Supplmentary Material, VSP2 expression was activated after primed by a set of NaCl at different concentrations (0, 50, 100, 150 mM). In this study, the defensive performance including resistance against herbivory and VSP2 expression in wild-type (Col-0), aos and myc2 backgrounds was investigated. To study the physiological effects of priming, the total glucosinolates (GSL) level and expression of biosynthesis genes SUR1 and FMO-OX3 were profiled. In addition, ROS accumulation and gene expression involved in ROS biogenesis and scavenging were investigated. Collectively, the results showed that salt-priming during seedling stage increased ROS level and GSL content even in adult plants. Salt-priming also mounted long-lasting defense against Spodoptera litura (Fabricius) larvae in adult plants, which was probably independent on MYC2- and AOS-controlled JA signaling pathway.

Materials and methods

Plant materials and priming treatment

Arabidopsis thaliana wild-type Col-0 and two knock-out mutants of critical genes in jasmonate pathway (aos, TAIR stock number: CS6149; myc2, TAIR stock number: SALK_083483) were used in the experiments. The block of JA biosynthesis in aos was reported²² and the knock-out of MYC2 was demonstrated in the supplementary materials (Figure S2 in Supplmentary Material; Table S1 in Supplmentary Material). The seeds were soaked in 75% alcohol for 10 min and rinsed three times with sterilized water. Then the surface-sterilized seeds were laid onto the MS medium supplemented with 100 mM NaCl as priming treatment (primed) or without NaCl as the control (non-primed). After stratified for 72 h at 4°C, the petri dishes with seeds were transferred to a walk-in growth chamber. The 10-day-old primed and non-primed seedlings were transplanted into autoclaved soil (Pindstrup^TM, Denmark). Unless stated otherwise, the grown conditions were 23 ± 1°C, 65 ± 5% relative humidity, light intensity of 180 μM photons m⁻²s⁻¹ and a 16/8 h photoperiod (light/dark). Three weeks after transplanted, eight-rosette-leave plants from primed and non-primed seedlings were subjected to subsequent assays.

Bioassay of resistance to herbivory

The eggs of S. litura (Fabricius), a herbivore causing severe defoliation of host plants,²⁷ were purchased (Baiyun Industrial Co., Ltd, Jiyuan, Henan, China) and reared on artificial diet under a 12 h light/12 h dark regime at 25 ± 1°C with 65 ± 5% relative humidity. For the larval feeding assay, two second-instar larvae were transferred onto the leaves of primed or non-primed plants three weeks after transplantation. Four days after infestation, each larva was weighed and the degree of foliar damage was analyzed by the software ImagJ.²⁸ The ion leakage of leaves after damage was measured by an electrolytic conductivity meter (AR8011, SmartSensor^TM, Dongguan, China) following the procedures with minor modifications.²⁹ Briefly, 0.5 g rosette leaves were collected and immersed in 10 ml of de-ionized water for 5 min. The water was subjected to electrical conductivity measurement. The values of the conductivity obtained from the sensor of the electrical conductivity meter were recorded and directly used to indicate the electrolyte leakage in damaged leaves.

Gene expression analyses by RT-qPCR

Approximately 0.1 g leaf tissue was subjected to total RNA isolation using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. After treated with RNase-Free DNase (Promega) to remove any DNA contamination, 2 μg total RNA was converted into cDNA using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific). Three technical replicates of qPCR were performed on a CFX96 Real-Time PCR System C1000 Touch Thermal Cycler (Bio-RAD) using the PowerUp^TM SYBR^TM Green Master mix (Applied Biosystems) according to the manufacturer’s instructions (http://thermofisher.com/support). Two-step PCR was carried out with 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 60 s following an initial denaturation of 2 min at 95°C. This program was followed by a melting curve analysis. The relative expression level of each gene was calculated by the ΔΔCt method using ACTIN2 (At3g18780) as internal reference genes.^30,31 The relative gene expression level was averaged over three biological replicates. Data was analyzed using the Student’s t test. The qPCR primer sequences of all analyzed genes are available as supporting information in Table S2 in Supplmentary Material.

Histochemical analysis of reactive oxygen species

Leaves from primed and non-primed Arabidopsis were collected and subjected to 3,3′-Diaminobenzidine (DAB) staining to visualize the accumulation of hydrogen peroxide (H₂O₂). After incubation, the stained samples were boiled for 10 min and bleached in fresh bleaching solution (ethanol/acetic acid/glycerol = 3:1:1, v/v/v). The stained samples were observed under stereoscopic microscope (Nikon stereoscope SMZ1000) or recorded by photographing. H₂O₂ content in leaves of primed or nonprime plants were quantified as described³² with a spectrometer (NanoDrop 2000, Thermo Scientific^TM).

Measurement of total glucosinolates by high-performance liquid chromatography (HPLC)

The measurement of total glucosinolates was modified based on the published procedures.³³ Briefly, 200 mg leaves from eight-rosette-leave plants were ground into fine powder then extracted with 2 ml of boiling 70% methanol for about 5 min. The mixture was then centrifuged at 12,000× g at 4°C for 15 min. The supernatant was collected and passed through 0.22 μM syringe filters and then loaded onto an anion-exchange column (DEAE-Sephadex A-25) treated overnight with aryl-sulfatase to convert the glucosinolates to their desulfated derivatives. After the conversion procedure, the desulfated glucosinolates were then eluted from the column with 70% (v/v) methanol. The eluent was evaporated to dryness under nitrogen at 45°C and resuspended in water. The desulfated glucosinolates were analyzed by high-performance liquid chromatography (HPLC) with the Agilent Infinity 1260 platform. The HPLC parameters were as follows: column, DiKMA C18 (Diomosil C18, 5 μm, 250 mm, 4.5 mm); mobile phase flow rate, 1 mL/min. Mobile phase parameters were programmed as follows: a linear gradient from 1.25% to 5% acetonitrile (v/v) over 5 min, a linear gradient from 7% to 8% acetonitrile (v/v) over the 2 min, a linear gradient from 8% to 30% acetonitrile (v/v) for 10 min, a linear gradient from 30% to 92% acetonitrile (v/v) over 3 min, constant at 92% acetonitrile (v/v) for 5 min followed by a linear gradient from 92% to 1.25% acetonitrile (v/v) over 1.5 min, then constant at 1.25% acetonitrile (v/v) for 5 min. Desulfated GSL was detected at A229 nm in a HPLC-DAD system. The GSL was quantified by comparison with 2-propenyl glucosinolate (sinigrin) as an internal standard. All tests were replicated three times.

Statistical analysis

Data were averaged from at least three independent biological replicates, and the data on various parameters were subjected to statistical analysis using GraphPad Prism 7 (https://www.graphpad.com). A one-way analysis of variance (ANOVA) was performed, and differences in means were separated using Dunnett’s multiple comparison test at a 1% level of significance. All data are presented as mean ± standard error.

Results

Salt-priming enhanced defense against larval feeding in adult plants

The leaf damage degree caused by larval feeding of primed plants was significantly lower compared to that of non-primed plants (P < .01) (Figure1a,b). The dry weight of larvae feeding on primed plants was decreased by 32% compared to that of larvae feeding on non-primed plants (Figure 1c), suggesting that the growth of larvae feeding on the plants primed by NaCl was inhibited. In the foliar ion leakage assay after feeding, the leakage in primed plants was 27.1% lower compared to that of non-primed plants (Figure S3 in Supplementary Material). The results showed that NaCl-induced priming could effectively protect Arabidopsis plants against S. litura. Previous exposure to NaCl imparted long-lasting defense ability even after Arabidopsis plants were transplanted into a stress-free environment.

Figure 1. Sodium chloride primes Arabidopsis plants against Spodoptera litura larval feeding.

Arabidopsis eight-rosette-leave plants were fed by second instar larvae of Spodoptera litura (Fabricius). (a) Primed plants and non-primed plants on the fourth day after infestation. (b) Rosette leaf damage caused by larval feeding in primed plants and non-primed plants. (c) Larval growth assessment after feeding on primed plants and non-primed plants. The scale bar represents 5 mm. Ten larvae were used to calculate mean values and standard deviations. Feeding assay was replicated above 3 times. Error bars indicate the standard deviation. ** differences are highly significant (P < .01) by Dunnett’s multiple comparison test. dai: days after infection by larvae. NP: non-primed plants; P: primed plants.

The relationship between NaCl-elicited defense priming and JA pathway

It is known that JA biosynthesis and JA signaling pathway play important roles in plant resistance to insect feeding.^19–21 To test whether an intact JA pathway is required for NaCl-primed defense against S. litura, the defensive performance against herbivory in aos and myc2 mutants was investigated. Compared to the non-primed plants, leaf damage caused by feeding was significantly decreased in plants that underwent salt-priming at their seedling stage (Figure 2a, b; Figure S3 in Supplmentary Material). Even in the aos and myc2 mutant plants, the defoliation degree was also significantly decreased by salt-priming. Bioassay results showed the growth of larvae was also effectively inhibited after feeding on primed plants of Col-0, aos or myc2 plants despite the different genetic backgrounds (Figure 2c, d). In addition, the expression levels of the VSP2 marker gene in primed plants increased by above 700% compared with that in plants from non-primed seedlings in both wild-type and mutant back grounds (Figure 2e). The results suggested that NaCl-induced defense priming against larval feeding is probably independent of MYC2- and AOS-controlled branches in the JA pathway.

Figure 2. Primed defense responses of Arabidopsis aos and myc2 mutants.

Arabidopsis eight-rosette-leave plants were fed by second instar larvae of Spodoptera litura (Fabricius). (a) Primed plants and non-primed plants on the fourth day after infestation. (b) Rosette leaf damage measurement by ImageJ. (c) Larvae on the fourth day after infestation; the scale bar represents 5 mm. (d) Larval growth assessment after feeding on primed plants and non-primed plants. Ten larvae were used to calculate mean values and standard deviations. Feeding assay was replicated above 3 times. (e) Relative expression levels of VSP2 in primed plants and non-primed plants in wild type Col-0 and two mutants, aos and myc2. Error bars indicate the standard deviation. ** differences are highly significant (P< .01) by Dunnett’s multiple comparison test. dai: days after infection by larvae. NP: non-primed plants; P: primed plants.

ROS and GSL level was increased by salt-priming

ROS accumulation is a common feature of many plant defense responses,³⁴ and glucosinolates (GSL) are a class of defensive secondary metabolites.^35,36 In this respect, salt-priming induced ROS and total GSL levels in Col-0 as well as expression of the genes involved in the relevant metabolic processes were investigated. As visualized by DAB staining and quantified with KI, respectively, primed plants accumulated more hydrogen peroxide (H₂O₂) compared to non-primed plants at seedling stage. Interestingly, after transplanted into a stress-free environment, H₂O₂ level persisted in eight-rosette-leave stage plants, which might be indicative of defensive priming state (Figure 3a, b). RT-qPCR results showed that the expression of RBOHA, RBOHB and RBOHC related to ROS production was increased by more than 30% and the expression levels of genes SOD2 and CAT1 related to ROS scavenging also increased by more than 100% (Figure 3c), which suggested ROS synthesis and degradation might be dynamically regulated and the maintaining the intracellular ROS homeostasis to support its defensive role and avoid cellular toxicity was also necessary. The amount of GSL was increased by 31% by salt-priming. The expression level of key genes SUR1 and FMOGS-OX3 related to GSL synthesis in plants undergoing salt-priming increased significantly as compared to that in non-primed plants (Figure 3d, e), suggesting that salt-priming modulated defensiveness in primed plants by maintaining higher GSL level.

Figure 3. ROS and GSL in non-primed and primed plants.

(a) DAB staining of leaves from Arabidopsis seedlings and mature plants with (P) or without (NP) 100 mM NaCl for 10 days; DAB staining of non-primed or primed plants at eight-rosette-leave stage. The scale bar represents 1 mm. (b) H₂O₂ content in non-primed plants. (c) Relative expression levels of RBOHA, RBOHB, RBOHC, SOD2 and CAT1 in non-primed and primed Arbidopsis at eight-rosette-leave stage. (d) Total glucosinolate content in Arabidopsis at eight-rosette-leave satge. (e) The expression of key genes SUR1 and FMO (FMOGS-OX3) related to glucosinolate synthesis in leaves of non-primed and primed plants at eight-rosette-leave stage. All tests were replicated three times. Error bars indicate the standard deviation. **differences are significant (P < .01) by Dunnett’s multiple comparison test.

Discussion

Priming has been well documented and proposed as an important strategy to increase the vitality of plants under adverse conditions.^2–16 Several studies suggested that NaCl as a priming agent could effectively enhance plant resistance to abiotic stresses.^25–29 Owing to its ionic properties, NaCl is commonly regarded as a hazardous factor³⁷ causing salinity stress and less used as a priming agent for protecting plants against impending biotic/abiotic stresses.

Here in this study, priming induced by NaCl effectively enhanced defense against larval feeding (Figure 1), activated ROS accumulation and increased the glucosinolates content (Figure 3). ROS is a universal cellular signal in many organisms while glucosinolates are known to be exclusively produced by Brassicaceae plants. Genes as ROS producers including RBOHA (AT5G07390), RBOHB (AT1G09090) and RBOHC (AT5G47910)^38,39 and scavengers including SOD2 (AT2G28190) and CAT1 (AT1G20630)^40,41 were both upregulated by salt-priming, suggesting even though the ROS level was elevated, the burst of ROS was monitored to avoid excessive cytotoxicity. Expression of SUR1 (AT2G20610) and FMOGS-OX3 (AT1G62560) was upregulated in primed plants, which might be causative of the increased GSL content regarding their important roles in GSL biosynthesis³⁵. Both ROS burst and glucosinolates accumulation are believed to be representative of defensive behaviors induced by biotic stresses.^36,41 Accumulation of ROS induced expression of genes involved in glucosinolate biogenesis⁴². In return, breakdown products of glucosinolates reduced the level of ROS and programmed cell death in Arabidopsis⁴³. In another report, breakdown products of glucosinolates increased peroxidase-mediated reactive oxygen species production⁴⁴. According to our results, ROS and GSL accumulation were simultaneously triggered by salt-priming. Due to the antioxidant and anti-insect roles of breakdown products of glucossinolate, glucosinolates might act as an intermediator to balance the defense and cytotoxicity. Thus, it is likely that ROS and glucosinolates are not absolutely independent but instead, their might act synergistically or antagonistically in certain situations.

Previous studies have shown that the JA signaling pathway plays a key role in plant resistance to insect feeding, and the mutation of key genes in the pathway will lead to impairment of JA-induced defense responses.^{19–26,45–47} However, neither the ability to resist insect feeding nor VSP2 gene expression levels were impaired in primed aos or myc2 mutant plants compared to the primed wild-type plants (Figure 2), suggesting priming-induced resistance to herbivory and elevated expression of VSP2 might be JA-independent. VSP2 has been reported to be regulated by MYC2-branch and directly regulated by MYC2 in the JA signaling pathway. However, gene expression in eukaryotes is often regulated by multiple factors which increases the plasticity of organisms in responses to changing environments. In JA-signaling pathway, MYC2 binds to G-box of target genes including VSP2.²⁵ In silico analysis reveals that the promoter of VSP2 is enriched with W-box, ABRE, and as-1 motifs⁴⁸ in addition to G-box, which are extensively involved in responses to both biotic and abiotic stresses. W-box is specific recognized by WRKY transcription factors and ABRE is responsive to ABA signaling. TGA members binds to as-1 motif to activate gene transcription. These cis-elements probably equip VSP2 with anchoring sites for other priming-induced transcription factors. Those transcription factors, which are JA-independent, might be involved in activation of VSP2 during the process of priming.

Our results indicated that salt-priming treatment at seedling stage enhanced adult plants’ defense responses against herbivory. The previously primed plants showed higher expression of VSP2 in response to scissor-clipping as a mimic of chewing herbivores (Figure S4 in Supplmentary Material). In response to wounding, the VSP2 expression level of primed plants was 79.7% higher than that of non-primed plants. The augmented VSP2 expression might improve plant performance in the battle with herbivores. Based on our results, NaCl-priming barely hindered Arabidopsis growth after germination. Instead, the priming treatment enhanced Arabidopsis growth after transplanted into stress-free environment, which was indicated by the increased biomass and rosette leaf size (Figure S5 in Supplmentary Material). Considering the fact that hyperactive defense often results in inhibited growth⁴⁹ and JA is a potent inhibitor of plant growth,^50,51 the primed defense by NaCl seems to be a leverage to improve plant performance under attack without the penalty of JA-mediated growth inhibition, which might be an additional bonus for increasing the yield of crop vegetative organs. Presumably the salt-priming could be regarded as a mild biotic stress, it’s surprising that Arabidopsis seedlings memorized such a “lesson” and activated defense responses even the environmental factors have been reset after the transplantation. In this respect, we proposed that the seedling priming induced by NaCl should have a promising prospect in agronomy, especially where seedlings were transplanted after germination and incubation artificially.

Author’s contributions

Mu Xiao conducted the experiments of seedling priming, bioassay, RT-qPCR, secondary metabolite measurements and data processing. Rong Liu was responsible for rearing the insects and verifying knock-out of MYC2. Ying Ruan and Chunlin Lin designed the experiments and drafted the manuscript. All authors participated in the manuscript revision.

Disclosure statement

No potential conflicts of interest were disclosed.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

This work was supported by the National Center for Research and Development [grant number 2016YFD0100202] and Education Department of Hunan Province [grant number 17A101], [grant number 17K045].