Viral walk: using ROS to explain CoRSV systemicity in Chenopodium quinoa Willd. at high temperature

Viral walk: using ROS to explain CoRSV systemicity in Chenopodium quinoa Willd. at high temperature | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Viral walk: using ROS to explain CoRSV systemicity in Chenopodium quinoa Willd. at high temperature Guilherme Holanda, Antonia Thalyta Lopes Silveira, José Manoel Ferreira de Lima Cruz, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5936731/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Sep, 2025 Read the published version in European Journal of Plant Pathology → Version 1 posted 6 You are reading this latest preprint version Abstract Dichorhavirus coffeae (CoRSV) can cause local or systemic symptoms in Chenopodium quinoa , depending on the temperature at which the plants are kept after mechanical inoculation. What causes this change in movement is still unknown. Increase in temperature and the presence of viruses can alter reactive oxygen species (ROS), which may be a means of explaining what enables systemic movement. The levels of hydrogen peroxide (H 2 O 2 ), antioxidants enzymes (superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT)), and malondialdehyde (MDA) were investigated in C. quinoa plants inoculated with CoRSV and kept under two temperature conditions. Six treatments were used: control (T1 and T4), inoculation with phosphate buffer (T2 and T5) and inoculation with CoRSV (T3 and T6). After inoculation, plants from treatments T1, T2, and T3 were kept in a greenhouse at an average temperature of 25°C, and plants from treatments T4, T5, and T6 were kept in a growth chamber at an average temperature of 28°C. Leaf analyses were performed at 0, 3, and 5 days after inoculation (DAI). SOD levels remained high in all treatments. However, plants kept at 25°C showed higher levels of H 2 O 2 and APX activity. Plants with viruses kept at 28°C showed reduced H 2 O 2 , APX, and CAT levels at 5 DAI, and high MDA levels. The high amount of MDA present in these plants may indicate that ROS escapes the antioxidant system and causes structural damage in C. quinoa , thus facilitating the systemic movement of CoRSV. Reactive oxygen species Coffee ringspot virus Dichorhavirus coffeae Viral movement Antioxidant enzymes Figures Figure 1 Figure 2 Figure 3 Introduction Chenopodium quinoa Willd., in addition to being considered a superfood (Graf et al., 2015 ) and one of the most important pseudocereals in the world (FAO, 2013 ), has been widely used as an indicator plant for viral infections, such as coffee ringspot virus ( Dichorhavirus coffeae , CoRSV) (Chagas et al., 1981 ; Ramalho et al., 2016 ). Unlike what occurs in coffee plants, where CoRSV causes only local lesions at the vector's feeding site, this virus can systemically infect C. quinoa plants if they are maintained at 28°C for five days after inoculation. Conversely, if after mechanical inoculation C. quinoa plants are kept at milder temperatures, around 25°C, only local infection occurs, demonstrating a temperature-dependent change in the virus-plant interaction (Ramalho et al., 2014 ). To date, the possible causes of this change in CoRSV movement within C. quinoa plants under different temperatures have not been investigated. D. coffeae belongs to the order Mononegavirales , family Rhabdoviridae (ICTV, 2025 ). It has enveloped bacilliform particles (35–40 nm x 100–140 nm), that are found in the nucleus and perinuclear spaces of infected cells (Chagas et al., 2003 ). The genome is bipartite, consisting of single-stranded negative-sense RNA, and encodes six ORFs: nucleocapsid (N), phosphoprotein (P), possible cell-to-cell movement protein (Y), matrix protein (M), glycoprotein (G) and RNA polymerase (L), arranged in RNA1 3'-N-P-Y-M-G-5' and in RNA2 3'-L-5' (Ramalho et al., 2014 ). The virus is found in plants of the genus Coffea and secondary hosts (Kitajima et al., 2011 ; Nunes et al., 2022 ) and field transmission occurs via the mites Brevipalpus phoenicis (Geijskes) sensu lato (Acari: Tenuipalpidae) (Beard et al., 2015 ; Chagas et al., 2003 ). Plants can respond to abiotic and biotic stress conditions, such as elevated temperature and virus invasion, through the production of reactive oxygen species (ROS) (Yang et al., 2024 ). With regard to viruses, the increase in ROS occurs due to immune responses induced by pattern-triggered immunity (PTI) or effector-triggered immunity (ETI) resulting in activation of metabolic pathways leading in programmed cell death (PCD) and the formation of localized lesions (Daudi et al., 2012 ; Xu et al., 2024 ). ROS are generated from molecular oxygen (O 2 ), which donates an electron to a receptor molecule, forming superoxide anion (O 2 − ). The enzyme superoxide dismutase (SOD) dismutates O 2 − into hydrogen peroxide (H 2 O 2 ) which can subsequently be converted into water (H 2 O) and oxygen (O 2 ) by the enzymes ascorbate peroxidase (APX) and catalase (CAT). In addition to O 2 − and H 2 O 2 , the hydroxyl radical (OH − ) also acts as ROS (Mittler et al., 2022 ). Oxidative stress can be assessed through lipid peroxidation levels, measured by malondialdehyde (MDA) content, the accumulation of H 2 O 2 and the balance of antioxidant enzymes in the system, which indirectly indicate O 2 − and H 2 O 2 . (Hakmaoui et al., 2012 ). Low to moderate ROS levels benefit the plant acting as elicitors against biotic and abiotic stress. However, high ROS levels, although effective in hypersensitivity response (HR), can cause oxidative damage, disrupt cellular functions, and facilitate viral infection and movement (Xu et al., 2024 ). The ability of viruses to establish systemic infection in plants relies on their capacity to replicate within the initially infected cells, move cell-to-cell via plasmodesmata, and reach the vascular tissue, where they are translocated throughout the plant (Kim et al., 2012 ). If viruses fail to complete this final stage of movement, the infection remains localized to a small cluster of neighboring cells (Wang, 2021 ). Environmental conditions before, during and after infection directly influence not only the progression of colonization (Scandolera et al., 2024 ), but also viral transmission, replication translocation within the plant and host susceptibility (Garcı́a-Castillo et al., 2001 ; Meziadi et al., 2021 ). Plants with elevated ROS levels may exhibit damaged cell walls and membranes, which can facilitate viral movement, as the plasmodesmata channels previously restricting this movement become compromised (Hernández et al., 2016 ; Sahu et al., 2022 ). The present study aimed to investigate the levels of reactive oxygen species and antioxidant enzymes, as well as malondialdehyde levels, in C. quinoa plants inoculated with CoRSV and maintained under two different temperatures conditions. Materials and methods Experimental design, plants material, treatments and inoculation procedure Healthy Chenopodium quinoa plants, 15 days post-sowing, were used for the experiments. C. quinoa plants infected with CoRSV served as the inoculum source. The treatments applied in the study are detailed in Table 1 . Table 1 – Description of treatments applied in the experiment with Chenopodium quinoa plants maintained for five days in two different temperatures conditions Treatments Details of treatments Environment (average temperature) T1 Control Greenhouse (25 ºC) T2 C. quinoa inoculated with phosphate buffer Greenhouse (25 ºC) T3 C. quinoa inoculated with CoRSV Greenhouse (25 ºC) T4 Control Growth chamber (28 ºC) T5 C. quinoa inoculated with phosphate buffer Growth chamber (28 ºC) T6 C. quinoa inoculated with CoRSV Growth chamber (28 ºC) In the mechanical inoculation procedures, the plants were first sprayed with carborundum 600 mesh. The extract was then prepared by macerating C. quinoa tissues in a phosphate buffer solution with a final concentration of 0.01 molar (M), supplemented with 0.01 M sodium sulfite at the time of use, in the ratio of 10 ml of buffer for 1 g of plant tissue. In treatments T1 and T4, the plants did not receive any interference. In treatments T2 and T5, inoculation was performed only with phosphate buffer, without plant material. In treatments T3 and T6, the plants were inoculated with material containing CoRSV. The plants were maintained in a greenhouse or growth chamber (Table 1 ) for 5 days after inoculation. The greenhouse and growth chamber had an average temperature of 25°C and 28°C, respectively. Fresh leaf samples were collected at 0, 3 and 5 days after inoculation (DAI). At time 0, collection was performed immediately after the inoculation procedure on the leaf blade in treatments T2, T3, T5 and T6, and for plants in treatments T1 and T4, collection was varried out without inoculation. The collected material was wrapped in aluminum foil, stored in liquid nitrogen (-80 ºC), and then transferred to an ultrafreezer (-80 ºC) until it was used for enzymatic analysis. Superoxide dismutase, catalase and ascorbate peroxidase analyses For the extraction of antioxidant enzymes, 200 mg of the plant material was macerated in liquid nitrogen with 10 mg of polyvinylpolypyrrolidone (PVPP) and homogenized with 1.5 mL of the extraction buffer (100 mM phosphate buffer pH 7.8, 0.1 mM EDTA and 10 mM ascorbic acid). The extract obtained was centrifuged at 12,000 rpm for 10 minutes at 4 ºC. The supernatant was collected and used in the enzymatic analyses performed below as Biemelt; Keetman; Albrecht, ( 1998 ). To determine superoxide dismutase (SOD) activity, 10 µL of the supernatant from each sample was mixed with 190 µL of the buffer solution (50 mM potassium phosphate, pH 7.8, 14 mM methionine, 0.1 mM EDTA, 75 µL of NBT, and 2 µM riboflavin). The samples were incubated in ELISA microtiter plates under a 20 W fluorescent lamp for 7 minutes, then read at 560 nm using a spectrophotometer. SOD activity was assessed based on the enzyme's ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT), as described by Giannopolitis; Ries ( 1977a , b ). The SOD content was calculated as the amount of enzyme required to inhibit 50% of the NBT reduction rate, and the result was expressed in U SOD min − 1 g − 1 of fresh weight (FW) Catalase (CAT) activity was measured by adding 162 µL of a solution composed of 100 mM potassium phosphate, containing 9 µL of 240 mM hydrogen peroxide, to 10 µL of the supernatant obtained previously. CAT activity was determined by the consumption of hydrogen peroxide, measured by the decrease in the absorbance of the extract at 240 nm at 15-second intervals over a period of 3 minutes (Havir e Mchal, 1987). The enzyme activity was defined as the amount of enzyme require to decompose 1 µM.min − 1 of H 2 O 2 . The result obtained was expressed as nmol H 2 O 2 min − 1 g − 1 FW. The quantification of peroxidase (APX) activity was performed using 9 µL of the previously obtained supernatant and 162 µL of a solution composed of 100 mM potassium phosphate and 0.5 mM ascorbic, acid preheated to 30 ºC. Then, 9 µL of 2 mM hydrogen peroxide was added, and the absorbance was measured in a spectrophotometer at 290 nm. APX activity was determined by the reduction in the absorbance of ascorbate (ε = 2.8 mM − 1 cm − 1 ) at 15-second intervals over a period of 3 minutes (Nakano & Asada, 1981 ). One unit of APX was defined as the amount of enzyme that oxidizes one µmol.min − 1 of ascorbic acid. The result was obtained in nmol ASA g − 1 FW − 1 min − 1 . Quantification of lipid peroxidation and hydrogen peroxide Lipid peroxidation and hydrogen peroxide (H 2 O 2 ) were measured using the method described by Buege e Aust (1978), as follows: 200 mg of plant material were macerated in liquid nitrogen and, to the powder obtained, 10 mg of PVPP and 1.5 mL of trichloroacetic acid (TCA) were added and the extract was incubated for 15 minutes and centrifuged at 12,000 rpm for 15 minutes at 4 ºC. The supernatant was collected and used for the following analyses. In the determination of lipid peroxidation, a 125 µL aliquot of the supernatant was mixed with 250 µL of the solution containing 10% TCA and 0.5% thiobarbituric acid (TBA). This mixture was heated at 95 ºC for 30 minutes and then immediately transferred to ice. The absorbance of this solution was read at 535 nm and 600 nm. The malondialdehyde (MDA) content was quantified as proposed by Buege e Aust (1978). The amount of MDA was calculated using the extraction coefficient 155 mM − 1 cm − 1 and the final result expressed in nmol MDA g − 1 FW. The H 2 O 2 measurement was performed according to the method proposed by Velikova; Yordanov; Edreva ( 2000 ). A 45 µL aliquot of the previously obtained supernatant was mixed with 135 µL of a solution containing 45 µL of 10 mM potassium phosphate (pH 7) and 90 µL of 1 M potassium iodide. The absorbance was measured in a spectrophotometer at a wavelength of 390 nm with the final result expressed in nmol H 2 O 2 mg − 1 FW. To generate the standard curve, the absorbance of 45 µL of the solution was measured at six different concentrations of H 2 O 2 : 0, 5, 15, 25, 35 and 45 µmol. Using the absorbance values ​​of the defined concentrations, the equation was established to quantify the hydrogen peroxide content in the samples. Confirmation of the presence of CoRSV For cDNA synthesis by reverse transcriptase (Sigma-Aldrich), 1 µL of total RNA (500 ng) previously extracted from the plant, 1 µL of random primers at 10 pM, 1 µL of dNTPs (10 mM) and 7 µL of ultrapure DEPC-treated water were initially mixed. This mixture was incubated for 10 minutes at 70 ºC, then immediately transferred to an ice container. In a subsequent step, 2 µL of 10x enzyme buffer (Sigma-Aldrich), 7 µL of ultrapure water and 1 µL of the MMLV reverse transcriptase enzyme (Sigma-Aldrich) were added, followed by incubation for 50 minutes at 37 ºC and 10 min at 85 ºC. The PCR reaction was performed using 1 µL of cDNA, 2.5 µL of 10x Taq DNA polymerase buffer (Cellco), 1 µL of forward primer and 1 µL of reverse primer, both at 10 pM, 1 µL of 10 mM dNTP, 0.26 µL of Taq DNA polymerase enzyme (Cellco) and DEPC-treated ultrapure water to a final volume of 25 µL. Amplification was carried out with an initial denaturation step at 94 ºC for 1 minute followed by 35 cycles: 94 ºC for 45 seconds, 59 ºC for 30 seconds and 72 ºC for 1 minute, with a final extension at 72 ºC for 5 minutes. The specific primers used amplify the initial portion of the nucleocapsid gene (105F: 5’-ATGGCTAGGTATGCGGATGTTG-3’ and 813R: 5’-GACTCTCATCAGCAACCGCACAG-3’), producing an amplicon of 708 base pairs. PCR products were verified on a 1% agarose gel stained with Gel Red (Biotium), using the 1 kb ladder marker (Ludwig) as a reference. Experimental design and statistics analysis Three plants (biological triplicate) were used per treatment at each collection time, totaling nine samples. For the enzymatic analyses of SOD, APX, CAT, H 2 O 2 , and MDA, each biological replicate was performed in technical triplicate in ELISA microplates, totaling nine readings per sample. Three wells containing only water were included in each plate as blanks. Statistical analyses were performed using R software (R Core Team, 2021) and the results were subjected to the Shapiro-Wilk test for residual normality test and Bartlett's test for homogeneity of variances. Data that met the assumptions of normality and homoscedasticity were subjected to analysis of variance (ANOVA) using the F test (p ≤ 0.05), with means compared using the Scott-Knott test (p ≤ 0.05). Results There was no significant statistical difference in superoxide dismutase (SOD) activity among the treatments, except in the control at 25 ºC (T1) at 5 days after inoculation (DAI). An increase in SOD levels was observed in C. quinoa plants at 5 DAI in the T4 treatment (60.12 U SOD min − 1 g − 1 FW) when compared to T1 (46.05 U SOD min − 1 g − 1 FW) (Fig. 1 A). In the T1 treatment, SOD activity decreased over time (46.05 at 0 DAI, 43.77 at 3 DAI and 31.18 U SOD min − 1 g − 1 FW at 5 DAI). Hydrogen peroxide (H 2 O 2 ) levels in treatments T4 and T6 were lower at 5 DAI, 1030.57 and 1140.21 mmol H 2 O 2 mg − 1 FW, respectively (Fig. 1 B). In contrast, treatments maintained at 25 ºC (T1 to T3) exhibit higher values, above 1652.56 mmol H 2 O 2 mg − 1 FW at 5 DAI. Among treatments involving CoRSV, T3 maintained high H 2 O 2 levels, while T6 showed an increase at 3 DAI followed by a decrease at 5 DAI. Ascorbate peroxidase (APX) activity was notably high in T3 at 5 DAI (3.42 nmol ASA min − 1 g − 1 FW) (Fig. 1 C) correlating with elevated H 2 O 2 levels (Fig. 1 B). Conversely, T6 exhibited reduced APX activity (1.71 nmol ASA min⁻¹ g⁻¹ FW) due to lower H 2 O 2 levels. In the T1 treatment, APX activity remained high, exceeding 3.63 nmol ASA min⁻¹ g⁻¹ FW, indicating an active reduction of H 2 O 2 levels (Fig. 1 B). On the other hand, T4 showed a reduction in APX activity at 5 DAI (0.89 nmol ASA min⁻¹ g⁻¹ FW). Catalase (CAT) activity exhibited an increase in the T3 treatment at 3 and 5 DAI (Fig. 1 D), likely due to the rise in H 2 O 2 levels caused by virus infection, as similarly observed for APX activity (Fig. 1 C). An increase in CAT activity was also noted in T4, potentially as a response to elevated H 2 O 2 levels induced by temperature. Overall, CAT levels remained high in C. quinoa plants, irrespective of abrasive effects or virus presence, ranging from 0.67 nmol H 2 O 2 min − 1 g − 1 FW in T4 at 0 DAI to 1.39 nmol H 2 O 2 min − 1 g − 1 FW in T5 at 3 DAI. In T6, CAT levels were consistent, suggesting that APX might be the main contributor to antioxidant activity. The quantification of lipid peroxidation, measured via the MDA method, showed variations from 4.68 nmol MDA g − 1 FW in T3 at 3 DAI to 18.18 nmol MDA g − 1 FW in T6 at 3 DAI (Fig. 2 ). Among treatments, T6, where plants were inoculated with CoRSV and maintained at 28 ºC, exhibited the highest MDA levels, particularly at 3 and 5 DAI. Treatments maintained at 25 ºC, especially T4 and T6, displayed lower H 2 O 2 activity (Fig. 1 B) and APX activity (Fig. 1 C) but sustained high SOD levels (Fig. 1 A), suggesting superoxide anion as the oxidizing agent. The presence of CoRSV was confirmed in T3 and T6 at 10 DAI through electrophoresis gel analysis, with bands observed at the specific size of 708 base pairs (Fig. 3 ). Discussion In this study, variations in the levels of reactive oxygen species (ROS), antioxidant enzymes, and malondialdehyde (MDA) were observed in Chenopodium quinoa plants under different temperature conditions (25°C and 28°C post-inoculation) and treatments with and without inoculation with CoRSV. The ROS pathway in plants begins with the generation of superoxide anion (O 2 − ), which is dismutated into hydrogen peroxide (H 2 O 2 ) by superoxide dismutase (SOD) (Mittler et al., 2022 ). Comparing the controls in both environments (Fig. 1 A), SOD levels decreased in T1, suggesting that a 25°C environment induces less O 2 − production compared to the 28°C environment (T4). Treatments T1, T2, and T3, maintained at lower temperatures, efficiently converted O 2 − to H 2 O 2 , as indicated by the H 2 O 2 levels (Fig. 1 B). Conversely, treatments T4 and T6, exposed to higher temperatures, displayed elevated SOD levels (Fig. 1 A) but continued to exhibit low H 2 O 2 levels (Fig. 1 B). This trend aligns with the observed low levels of APX activity in these treatments (Fig. 1 C). In treatment T6, plants inoculated with CoRSV and kept at 28°C demonstrated high levels of O 2 − , indicating incomplete conversion to H 2 O 2 . The ambient temperature likely impeded the enzymatic conversion process. Consequently, the elevated O 2 − levels, compounded by viral infection, may have contributed to increased lipid peroxidation, quantified through malondialdehyde (MDA) levels (Fig. 2 ). MDA, a product of lipid peroxidation, is widely regarded as a reliable indicator of cell membrane degradation and a marker of oxidative stress (Abdelkhalek et al., 2022 ). In this study, it was observed (Fig. 2 ) that C. quinoa plants inoculated with CoRSV and maintained at a temperature of 28°C (T6) exhibited elevated MDA levels, reflecting significant cell membrane degradation. Previous studies by El-moshaty et al., ( 1993 ) and Hakmaoui et al., ( 2012 ) have demonstrated that higher O 2 − levels lead to increased lipid peroxidation, with subsequent MDA levels declining as tissue structure begins to collapse. The decrease in MDA levels observed in T6 at 5 DAI could be attributed to this structural breakdown, occurring after the peak activity noted at 3 DAI. Several studies corroborate these findings, indicating that the presence of viruses in plants contributes to heightened lipid peroxidation due to increased ROS production (Abdelkhalek et al., 2022 ; Pérez-Clemente et al., 2015 ; Sofy et al., 2021 ). The damage to the cellular structure of C. quinoa , as evidenced by the elevated MDA levels in the T6 condition (inoculation with CoRSV and maintained at 28°C) (Fig. 2 ), may play a crucial role in explaining the plant's capacity for long-distance viral movement. Lipid peroxidation damage can compromise the selectivity and stability of cellular membranes, promoting leakage and abnormal interactions (Kar, 2023). This compromised membrane integrity could facilitate the movement of viral proteins and genomes to neighboring cells (Alazem & Burch-Smith, 2024 ). Excessive O 2 − can lead to oxidation of the cell wall, making it more fragile and less restrictive to viral movement (Novaković et al., 2018 ). This could result in an increase in the size exclusion limit (SEL) of plasmodesmata, as seen in other plant-virus interactions (Zhou et al., 2019 ). The combination of damage to the cell wall and plasma membrane may enable the virus to more easily move through cells, shortening the distance to the plant's conducting vessels, where it can initiate systemic infection. Elevated levels of H 2 O 2 in plants, along with other associated changes, induce cell wall fortification and callose deposition (Alazem & Burch-Smith, 2024 ; Khan et al., 2023 ). These processes reduce the SEL of plasmodesmata and hinder viral movement through this pathway (Kumar & Dasgupta, 2021 ). Additionally, high H 2 O 2 levels are known to activate systemic acquired resistance (SAR) pathways (Jwa & Hwang, 2017 ). In C. quinoa plants maintained at 25°C, the initial oxidative response, coupled with cell wall strengthening due to increased H 2 O 2 levels (Fig. 1 B), and the low lipid peroxidation observed (Fig. 2 ), likely restrict viral movement to the local infection site. This suggests that, under these conditions, the host has not suffered significant structural damage that would allow for long-distance viral movement. In contrast, the situation is different for plants inoculated with CoRSV and kept at 28°C, as previously discussed. The higher temperatures seem to impair the plant's ability to mount an effective defense, facilitating viral spread. For CoRSV, there are several factors that favor its replication and movement at 28°C. It is suggested that the formation of viroplasm is temperature-dependent, with the higher temperature enabling greater viral content (M. Goodin, unpublished data). This results in increased viral accumulation and the development of systemic symptoms, as observed by Amari, Huang e Heinlein, (2021). Also at high temperature, the movement protein (MP) is more likely to dock at plasmodesma receptor sites or alter its accumulation site, shifting from the replication site to the plasmodesma (Heinlein, 2015 ). This modification can lead to an increase in SEL of plasmodesmata, facilitating viral movement through adjacent cells until it reaches the conducting vessels. Ultimately, this process leads to the appearance of systemic symptoms, highlighting the temperature's role in promoting CoRSV systemic spread. It was observed that CAT levels in the treatment with the virus at 25 ºC (T3) increased at 3 and 5 DAI, whereas in the treatment with CoRSV at 28 ºC (T6), these levels remained somewhat constant, as if CAT had not been activated (Fig. 1 D). Peroxidase levels were more variable (Fig. 1 C), with high activity at T3 at 5 DAI. Several factors may explain the behavior of these enzymes: i) the plant may maintain constant or reduced levels of catalase to keep H 2 O 2 levels high, thus assigning the dismutation function to APX (Ali et al., 2006 ; Riedle-Bauer, 2000 ); ii) despite the high rate of CAT, it has a lower affinity for H 2 O 2 than APX, which is why APX increases rapidly during pathogen infection (Mhamdi et al., 2010 ), while CAT increases almost linearly as H 2 O 2 levels rise (Mhamdi et al., 2010 , 2012 ); iii) the initial damage may have occurred in the chloroplasts, where APX activity is greater, and CAT is predominantly concentrated in peroxisomes (Sousa et al., 2019 ). Literature data demonstrate that during viral infection, CAT activity tends to decrease over time, while APX levels increase (Pérez-Clemente et al., 2015 ; Riedle-Bauer, 2000 ). C. quinoa plants infected with CoRSV exhibit the same symptom dynamics as those presented by carnation mottle virus (CarMV), a member of the Tombusviridae family. When kept at mild temperatures (25°C during the day and 18°C at night), only local symptoms appeared. In contrast, when exposed to high temperatures (between 35 and 40°C during the day and 12 to 15°C at night), virus movement within the plant was observed, accompanied by the onset of systemic symptoms. However, Garcı́a-Castillo et al., 2001 through immunohistochemical tests, reported that plants kept at both low and high temperatures had the same viral titer, with the pathogen reaching the conducting vessels in both conditions. The authors noted that plant defenses are activated only after the pathogen invades the vascular bundles, and that temperature facilitates a delay in the defense response. Since most plant viruses use the phloem to move throughout the plant, temperature is likely a critical factor in influencing this source-sink relationship (Nagamani et al., 2020 ). The growing conditions at 28°C may accelerate the translocation of photoassimilates, and in combination with cellular damage, facilitate the systemic movement of CoRSV. Future studies could explore whether the number of CoRSV copies varies with temperature and host, as observed by Nagamani et al. ( 2020 ). Conclusions C. quinoa plants kept in an environment with an average temperature of 28°C for five days after inoculation with CoRSV showed a reduction in hydrogen peroxide (H 2 O 2 ) accumulation, indicating that the plants are saturated with superoxide anion (O 2 − ). This reactive oxygen species can escape from the antioxidant system and lead to lipid peroxidation. Consequently, this damage may affect the cellular structure of the plant, facilitating the systemic movement of the virus. These factors could offer insights into the systemic movement of the virus at 28°C. Declarations Conflict of interest The authors declare that they do not have any actual or potential conflict of interest. Authors’ contributions All authors contributed to the study conception, design, data acquisition, data analysis and interpretation, as well as the drafting of the work. All authors read and approved the fnal manuscript. Acknowledgements The authors would like to thank the Brazilian National Council for Scientific and Technological Development (CNPq), the Coordination of Superior Level Staf Improvement (CAPES) and Foundation for Research Support of the State of Minas Gerais (FAPEMIG) for their support in this project. Data Availability The data supporting the findings of this study are available from the corresponding author upon request. References Abdelkhalek, A., El-Gendi, H., Al-Askar, A. A., Maresca, V., Moawad, H., Elsharkawy, M. M., Younes, H. A., & Behiry, S. I. (2022). 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Ameliorating the Adverse Effects of Tomato mosaic tobamovirus Infecting Tomato Plants in Egypt by Boosting Immunity in Tomato Plants Using Zinc Oxide Nanoparticles. Molecules , 26 (5), Artigo 5. https://doi.org/10.3390/molecules26051337 Sousa, R. H. V., Carvalho, F. E. L., Lima-Melo, Y., Alencar, V. T. C. B., Daloso, D. M., Margis-Pinheiro, M., Komatsu, S., & Silveira, J. A. G. (2019). Impairment of peroxisomal APX and CAT activities increases protection of photosynthesis under oxidative stress. Journal of Experimental Botany , 70 (2), 627–639. https://doi.org/10.1093/jxb/ery354 Velikova, V., Yordanov, I., & Edreva, A. (2000). Oxidative stress and some antioxidant systems in acid rain-treated bean plants: Protective role of exogenous polyamines. Plant Science , 151 (1), 59–66. https://doi.org/10.1016/S0168-9452(99)00197-1 Wang, A. (2021). Cell-to-cell movement of plant viruses via plasmodesmata: A current perspective on potyviruses. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5936731","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":412455524,"identity":"bbab4687-8b00-439e-abe8-bc7d53884be3","order_by":0,"name":"Guilherme Holanda","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-4501-1366","institution":"Universidade Federal de Lavras Departamento de Fitopatologia","correspondingAuthor":true,"prefix":"","firstName":"Guilherme","middleName":"","lastName":"Holanda","suffix":""},{"id":412455525,"identity":"0a333268-50bc-43d0-8b0a-17d24eae7adf","order_by":1,"name":"Antonia Thalyta Lopes Silveira","email":"","orcid":"","institution":"Universidade Federal de Lavras Departamento de Fitopatologia","correspondingAuthor":false,"prefix":"","firstName":"Antonia","middleName":"Thalyta Lopes","lastName":"Silveira","suffix":""},{"id":412455526,"identity":"ff166b16-48dc-4ffe-b868-821f3618cf49","order_by":2,"name":"José Manoel Ferreira de Lima Cruz","email":"","orcid":"","institution":"Universidade Federal de Lavras Departamento de Fitopatologia","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Manoel Ferreira de Lima","lastName":"Cruz","suffix":""},{"id":412455527,"identity":"fc714fe5-27f6-4af2-b0cf-23f610931211","order_by":3,"name":"Layla Victória da Silva Sousa","email":"","orcid":"","institution":"Universidade Federal de Lavras Departamento de Fitopatologia","correspondingAuthor":false,"prefix":"","firstName":"Layla","middleName":"Victória da Silva","lastName":"Sousa","suffix":""},{"id":412455528,"identity":"0779dea2-8be5-4f23-80cb-aec5e3c64d5e","order_by":4,"name":"Antonia dos Reis Figueira","email":"","orcid":"","institution":"Universidade Federal de Lavras Departamento de Fitopatologia","correspondingAuthor":false,"prefix":"","firstName":"Antonia","middleName":"dos Reis","lastName":"Figueira","suffix":""},{"id":412455529,"identity":"b31963af-9368-4e64-8723-94d0e4cf87b5","order_by":5,"name":"Wilson Vicente Souza Pereira","email":"","orcid":"","institution":"Universidade Federal do Piaui","correspondingAuthor":false,"prefix":"","firstName":"Wilson","middleName":"Vicente Souza","lastName":"Pereira","suffix":""},{"id":412455530,"identity":"a2e443d3-805c-4717-8dd3-1bb7a0db6e98","order_by":6,"name":"Heloisa Oliveira dos Santos","email":"","orcid":"","institution":"Universidade Federal de Lavras Departamento de Agricultura","correspondingAuthor":false,"prefix":"","firstName":"Heloisa","middleName":"Oliveira dos","lastName":"Santos","suffix":""},{"id":412455531,"identity":"51681f64-7521-4a23-bcb2-8c356eb63bb4","order_by":7,"name":"Claudine Marcia Carvalho","email":"","orcid":"","institution":"Universidade Federal de Lavras Departamento de Fitopatologia","correspondingAuthor":false,"prefix":"","firstName":"Claudine","middleName":"Marcia","lastName":"Carvalho","suffix":""}],"badges":[],"createdAt":"2025-01-31 13:45:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5936731/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5936731/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10658-025-03136-8","type":"published","date":"2025-09-26T15:57:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75881162,"identity":"38b4e618-27e6-42a7-8a99-4292c504cbab","added_by":"auto","created_at":"2025-02-10 08:35:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6839668,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of superoxide dismutase (SOD) (A), ascorbate peroxidase (APX) (B), catalase (CAT) (C), and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) (D) levels in \u003cem\u003eChenopodium quinoa\u003c/em\u003e plants subjected to different treatments and temperature conditions at 0, 3, and 5 days after inoculation (DAI). Treatments T1, T2, and T3 were maintained in a greenhouse with an average temperature of 25 ºC, while T4, T5, and T6 were kept in a growth chamber with an average temperature of 28 ºC. T1 and T4: controls without mechanical inoculation; T2 and T5: \u003cem\u003eC. quinoa\u003c/em\u003e mechanically inoculated with buffer only; T3 and T6: \u003cem\u003eC. quinoa\u003c/em\u003e mechanically inoculated with CoRSV. Means followed by the same lowercase letter between treatments and uppercase letter within treatments are statistically equal according to the Scott-Knott test (p≤ 0.05). Error bars represent the standard deviation\u003c/p\u003e","description":"","filename":"Fig1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5936731/v1/2f37b9a12fb2859a4257e604.jpg"},{"id":75882784,"identity":"4d766f5b-3e10-4b1a-b5c8-fbf39527325b","added_by":"auto","created_at":"2025-02-10 08:43:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2933111,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of malondialdehyde (MDA) levels in \u003cem\u003eChenopodium quinoa\u003c/em\u003e plants subjected to different treatments and temperature conditions at 0, 3, and 5 days after inoculation (DAI). Treatments T1, T2, and T3 were maintained in a greenhouse with an average temperature of 25 ºC, while T4, T5, and T6 were kept in a growth chamber with an average temperature of 28 ºC. T1 and T4: controls without mechanical inoculation; T2 and T5: \u003cem\u003eC. quinoa\u003c/em\u003e mechanically inoculated with buffer only; T3 and T6: \u003cem\u003eC. quinoa\u003c/em\u003e mechanically inoculated with CoRSV. Means followed by the same lowercase letter between treatments and uppercase letter within treatments are statistically equal according to the Scott-Knott test (p≤ 0.05). Error bars represent the standard deviation\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5936731/v1/2be0906a1e052e8aeb74429d.jpg"},{"id":75882785,"identity":"38bcfe52-20fc-42e0-bd5d-e861fe0d162f","added_by":"auto","created_at":"2025-02-10 08:43:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1510574,"visible":true,"origin":"","legend":"\u003cp\u003e1% agarose gel showing specific bands corresponding to the 708 bp amplicon of \u003cem\u003eDichorhavirus coffeae\u003c/em\u003e (CoRSV), RT-PCR products obtained with primers 105F/813R, 10 days after virus inoculation in \u003cem\u003eChenopodium quinoa\u003c/em\u003e. Treatments T1, T2, and T3 were maintained in a greenhouse with an average temperature of 25 ºC, while T4, T5, and T6 were kept in a growth chamber with an average temperature of 28 ºC. T1 and T4: controls without mechanical inoculation; T2 and T5: \u003cem\u003eC. quinoa\u003c/em\u003e mechanically inoculated with buffer only; T3 and T6: \u003cem\u003eC. quinoa\u003c/em\u003e mechanically inoculated with CoRSV\u003c/p\u003e","description":"","filename":"Fig3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5936731/v1/a60680c02838b52860310b12.jpg"},{"id":92430589,"identity":"d33aecfc-de43-43bb-9974-8ad3c24c8e1a","added_by":"auto","created_at":"2025-09-29 16:06:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11930266,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5936731/v1/f4f69ef0-f45d-444a-bbca-20ddec56109c.pdf"}],"financialInterests":"","formattedTitle":"Viral walk: using ROS to explain CoRSV systemicity in Chenopodium quinoa Willd. at high temperature","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eChenopodium quinoa\u003c/em\u003e Willd., in addition to being considered a superfood (Graf et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and one of the most important pseudocereals in the world (FAO, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), has been widely used as an indicator plant for viral infections, such as coffee ringspot virus (\u003cem\u003eDichorhavirus coffeae\u003c/em\u003e, CoRSV) (Chagas et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Ramalho et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Unlike what occurs in coffee plants, where CoRSV causes only local lesions at the vector's feeding site, this virus can systemically infect \u003cem\u003eC. quinoa\u003c/em\u003e plants if they are maintained at 28\u0026deg;C for five days after inoculation. Conversely, if after mechanical inoculation \u003cem\u003eC. quinoa\u003c/em\u003e plants are kept at milder temperatures, around 25\u0026deg;C, only local infection occurs, demonstrating a temperature-dependent change in the virus-plant interaction (Ramalho et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). To date, the possible causes of this change in CoRSV movement within \u003cem\u003eC. quinoa\u003c/em\u003e plants under different temperatures have not been investigated.\u003c/p\u003e \u003cp\u003e \u003cem\u003eD. coffeae\u003c/em\u003e belongs to the order \u003cem\u003eMononegavirales\u003c/em\u003e, family \u003cem\u003eRhabdoviridae\u003c/em\u003e (ICTV, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). It has enveloped bacilliform particles (35\u0026ndash;40 nm x 100\u0026ndash;140 nm), that are found in the nucleus and perinuclear spaces of infected cells (Chagas et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The genome is bipartite, consisting of single-stranded negative-sense RNA, and encodes six ORFs: nucleocapsid (N), phosphoprotein (P), possible cell-to-cell movement protein (Y), matrix protein (M), glycoprotein (G) and RNA polymerase (L), arranged in RNA1 3'-N-P-Y-M-G-5' and in RNA2 3'-L-5' (Ramalho et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The virus is found in plants of the genus \u003cem\u003eCoffea\u003c/em\u003e and secondary hosts (Kitajima et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Nunes et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and field transmission occurs via the mites \u003cem\u003eBrevipalpus phoenicis\u003c/em\u003e (Geijskes) \u003cem\u003esensu lato\u003c/em\u003e (Acari: Tenuipalpidae) (Beard et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chagas et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlants can respond to abiotic and biotic stress conditions, such as elevated temperature and virus invasion, through the production of reactive oxygen species (ROS) (Yang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). With regard to viruses, the increase in ROS occurs due to immune responses induced by pattern-triggered immunity (PTI) or effector-triggered immunity (ETI) resulting in activation of metabolic pathways leading in programmed cell death (PCD) and the formation of localized lesions (Daudi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). ROS are generated from molecular oxygen (O\u003csub\u003e2\u003c/sub\u003e), which donates an electron to a receptor molecule, forming superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e). The enzyme superoxide dismutase (SOD) dismutates O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e into hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) which can subsequently be converted into water (H\u003csub\u003e2\u003c/sub\u003eO) and oxygen (O\u003csub\u003e2\u003c/sub\u003e) by the enzymes ascorbate peroxidase (APX) and catalase (CAT). In addition to O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, the hydroxyl radical (OH\u003csup\u003e\u0026minus;\u003c/sup\u003e) also acts as ROS (Mittler et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Oxidative stress can be assessed through lipid peroxidation levels, measured by malondialdehyde (MDA) content, the accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and the balance of antioxidant enzymes in the system, which indirectly indicate O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. (Hakmaoui et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Low to moderate ROS levels benefit the plant acting as elicitors against biotic and abiotic stress. However, high ROS levels, although effective in hypersensitivity response (HR), can cause oxidative damage, disrupt cellular functions, and facilitate viral infection and movement (Xu et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe ability of viruses to establish systemic infection in plants relies on their capacity to replicate within the initially infected cells, move cell-to-cell via plasmodesmata, and reach the vascular tissue, where they are translocated throughout the plant (Kim et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). If viruses fail to complete this final stage of movement, the infection remains localized to a small cluster of neighboring cells (Wang, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Environmental conditions before, during and after infection directly influence not only the progression of colonization (Scandolera et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), but also viral transmission, replication translocation within the plant and host susceptibility (Garcı́a-Castillo et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Meziadi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Plants with elevated ROS levels may exhibit damaged cell walls and membranes, which can facilitate viral movement, as the plasmodesmata channels previously restricting this movement become compromised (Hern\u0026aacute;ndez et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sahu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present study aimed to investigate the levels of reactive oxygen species and antioxidant enzymes, as well as malondialdehyde levels, in \u003cem\u003eC. quinoa\u003c/em\u003e plants inoculated with CoRSV and maintained under two different temperatures conditions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eExperimental design, plants material, treatments and inoculation procedure\u003c/p\u003e \u003cp\u003eHealthy \u003cem\u003eChenopodium quinoa\u003c/em\u003e plants, 15 days post-sowing, were used for the experiments. \u003cem\u003eC. quinoa\u003c/em\u003e plants infected with CoRSV served as the inoculum source. The treatments applied in the study are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u0026ndash; Description of treatments applied in the experiment with \u003cem\u003eChenopodium quinoa\u003c/em\u003e plants maintained for five days in two different temperatures conditions\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDetails of treatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEnvironment (average temperature)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse (25 \u0026ordm;C)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC. quinoa\u003c/em\u003e inoculated with phosphate buffer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse (25 \u0026ordm;C)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC. quinoa\u003c/em\u003e inoculated with CoRSV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse (25 \u0026ordm;C)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrowth chamber (28 \u0026ordm;C)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC. quinoa\u003c/em\u003e inoculated with phosphate buffer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrowth chamber (28 \u0026ordm;C)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC. quinoa\u003c/em\u003e inoculated with CoRSV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrowth chamber (28 \u0026ordm;C)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn the mechanical inoculation procedures, the plants were first sprayed with carborundum 600 mesh. The extract was then prepared by macerating \u003cem\u003eC. quinoa\u003c/em\u003e tissues in a phosphate buffer solution with a final concentration of 0.01 molar (M), supplemented with 0.01 M sodium sulfite at the time of use, in the ratio of 10 ml of buffer for 1 g of plant tissue. In treatments T1 and T4, the plants did not receive any interference. In treatments T2 and T5, inoculation was performed only with phosphate buffer, without plant material. In treatments T3 and T6, the plants were inoculated with material containing CoRSV.\u003c/p\u003e \u003cp\u003eThe plants were maintained in a greenhouse or growth chamber (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) for 5 days after inoculation. The greenhouse and growth chamber had an average temperature of 25\u0026deg;C and 28\u0026deg;C, respectively.\u003c/p\u003e \u003cp\u003eFresh leaf samples were collected at 0, 3 and 5 days after inoculation (DAI). At time 0, collection was performed immediately after the inoculation procedure on the leaf blade in treatments T2, T3, T5 and T6, and for plants in treatments T1 and T4, collection was varried out without inoculation. The collected material was wrapped in aluminum foil, stored in liquid nitrogen (-80 \u0026ordm;C), and then transferred to an ultrafreezer (-80 \u0026ordm;C) until it was used for enzymatic analysis.\u003c/p\u003e \u003cp\u003eSuperoxide dismutase, catalase and ascorbate peroxidase analyses\u003c/p\u003e \u003cp\u003eFor the extraction of antioxidant enzymes, 200 mg of the plant material was macerated in liquid nitrogen with 10 mg of polyvinylpolypyrrolidone (PVPP) and homogenized with 1.5 mL of the extraction buffer (100 mM phosphate buffer pH 7.8, 0.1 mM EDTA and 10 mM ascorbic acid). The extract obtained was centrifuged at 12,000 rpm for 10 minutes at 4 \u0026ordm;C. The supernatant was collected and used in the enzymatic analyses performed below as Biemelt; Keetman; Albrecht, (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo determine superoxide dismutase (SOD) activity, 10 \u0026micro;L of the supernatant from each sample was mixed with 190 \u0026micro;L of the buffer solution (50 mM potassium phosphate, pH 7.8, 14 mM methionine, 0.1 mM EDTA, 75 \u0026micro;L of NBT, and 2 \u0026micro;M riboflavin). The samples were incubated in ELISA microtiter plates under a 20 W fluorescent lamp for 7 minutes, then read at 560 nm using a spectrophotometer. SOD activity was assessed based on the enzyme's ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT), as described by Giannopolitis; Ries (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1977a\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003eb\u003c/span\u003e). The SOD content was calculated as the amount of enzyme required to inhibit 50% of the NBT reduction rate, and the result was expressed in U SOD min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of fresh weight (FW)\u003c/p\u003e \u003cp\u003eCatalase (CAT) activity was measured by adding 162 \u0026micro;L of a solution composed of 100 mM potassium phosphate, containing 9 \u0026micro;L of 240 mM hydrogen peroxide, to 10 \u0026micro;L of the supernatant obtained previously. CAT activity was determined by the consumption of hydrogen peroxide, measured by the decrease in the absorbance of the extract at 240 nm at 15-second intervals over a period of 3 minutes (Havir e Mchal, 1987). The enzyme activity was defined as the amount of enzyme require to decompose 1 \u0026micro;M.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The result obtained was expressed as nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW.\u003c/p\u003e \u003cp\u003eThe quantification of peroxidase (APX) activity was performed using 9 \u0026micro;L of the previously obtained supernatant and 162 \u0026micro;L of a solution composed of 100 mM potassium phosphate and 0.5 mM ascorbic, acid preheated to 30 \u0026ordm;C. Then, 9 \u0026micro;L of 2 mM hydrogen peroxide was added, and the absorbance was measured in a spectrophotometer at 290 nm. APX activity was determined by the reduction in the absorbance of ascorbate (ε\u0026thinsp;=\u0026thinsp;2.8 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 15-second intervals over a period of 3 minutes (Nakano \u0026amp; Asada, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). One unit of APX was defined as the amount of enzyme that oxidizes one \u0026micro;mol.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of ascorbic acid. The result was obtained in nmol ASA g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eQuantification of lipid peroxidation and hydrogen peroxide\u003c/p\u003e \u003cp\u003eLipid peroxidation and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) were measured using the method described by Buege e Aust (1978), as follows: 200 mg of plant material were macerated in liquid nitrogen and, to the powder obtained, 10 mg of PVPP and 1.5 mL of trichloroacetic acid (TCA) were added and the extract was incubated for 15 minutes and centrifuged at 12,000 rpm for 15 minutes at 4 \u0026ordm;C. The supernatant was collected and used for the following analyses.\u003c/p\u003e \u003cp\u003eIn the determination of lipid peroxidation, a 125 \u0026micro;L aliquot of the supernatant was mixed with 250 \u0026micro;L of the solution containing 10% TCA and 0.5% thiobarbituric acid (TBA). This mixture was heated at 95 \u0026ordm;C for 30 minutes and then immediately transferred to ice. The absorbance of this solution was read at 535 nm and 600 nm. The malondialdehyde (MDA) content was quantified as proposed by Buege e Aust (1978). The amount of MDA was calculated using the extraction coefficient 155 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the final result expressed in nmol MDA g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW.\u003c/p\u003e \u003cp\u003eThe H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e measurement was performed according to the method proposed by Velikova; Yordanov; Edreva (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). A 45 \u0026micro;L aliquot of the previously obtained supernatant was mixed with 135 \u0026micro;L of a solution containing 45 \u0026micro;L of 10 mM potassium phosphate (pH 7) and 90 \u0026micro;L of 1 M potassium iodide. The absorbance was measured in a spectrophotometer at a wavelength of 390 nm with the final result expressed in nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW. To generate the standard curve, the absorbance of 45 \u0026micro;L of the solution was measured at six different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: 0, 5, 15, 25, 35 and 45 \u0026micro;mol. Using the absorbance values ​​of the defined concentrations, the equation was established to quantify the hydrogen peroxide content in the samples.\u003c/p\u003e \u003cp\u003eConfirmation of the presence of CoRSV\u003c/p\u003e \u003cp\u003eFor cDNA synthesis by reverse transcriptase (Sigma-Aldrich), 1 \u0026micro;L of total RNA (500 ng) previously extracted from the plant, 1 \u0026micro;L of random primers at 10 pM, 1 \u0026micro;L of dNTPs (10 mM) and 7 \u0026micro;L of ultrapure DEPC-treated water were initially mixed. This mixture was incubated for 10 minutes at 70 \u0026ordm;C, then immediately transferred to an ice container. In a subsequent step, 2 \u0026micro;L of 10x enzyme buffer (Sigma-Aldrich), 7 \u0026micro;L of ultrapure water and 1 \u0026micro;L of the MMLV reverse transcriptase enzyme (Sigma-Aldrich) were added, followed by incubation for 50 minutes at 37 \u0026ordm;C and 10 min at 85 \u0026ordm;C.\u003c/p\u003e \u003cp\u003eThe PCR reaction was performed using 1 \u0026micro;L of cDNA, 2.5 \u0026micro;L of 10x Taq DNA polymerase buffer (Cellco), 1 \u0026micro;L of forward primer and 1 \u0026micro;L of reverse primer, both at 10 pM, 1 \u0026micro;L of 10 mM dNTP, 0.26 \u0026micro;L of Taq DNA polymerase enzyme (Cellco) and DEPC-treated ultrapure water to a final volume of 25 \u0026micro;L. Amplification was carried out with an initial denaturation step at 94 \u0026ordm;C for 1 minute followed by 35 cycles: 94 \u0026ordm;C for 45 seconds, 59 \u0026ordm;C for 30 seconds and 72 \u0026ordm;C for 1 minute, with a final extension at 72 \u0026ordm;C for 5 minutes. The specific primers used amplify the initial portion of the nucleocapsid gene (105F: 5\u0026rsquo;-ATGGCTAGGTATGCGGATGTTG-3\u0026rsquo; and 813R: 5\u0026rsquo;-GACTCTCATCAGCAACCGCACAG-3\u0026rsquo;), producing an amplicon of 708 base pairs. PCR products were verified on a 1% agarose gel stained with Gel Red (Biotium), using the 1 kb ladder marker (Ludwig) as a reference.\u003c/p\u003e \u003cp\u003eExperimental design and statistics analysis\u003c/p\u003e \u003cp\u003eThree plants (biological triplicate) were used per treatment at each collection time, totaling nine samples. For the enzymatic analyses of SOD, APX, CAT, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and MDA, each biological replicate was performed in technical triplicate in ELISA microplates, totaling nine readings per sample. Three wells containing only water were included in each plate as blanks.\u003c/p\u003e \u003cp\u003eStatistical analyses were performed using R software (R Core Team, 2021) and the results were subjected to the Shapiro-Wilk test for residual normality test and Bartlett's test for homogeneity of variances. Data that met the assumptions of normality and homoscedasticity were subjected to analysis of variance (ANOVA) using the F test (p\u0026thinsp;\u0026le;\u0026thinsp;0.05), with means compared using the Scott-Knott test (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThere was no significant statistical difference in superoxide dismutase (SOD) activity among the treatments, except in the control at 25 \u0026ordm;C (T1) at 5 days after inoculation (DAI). An increase in SOD levels was observed in \u003cem\u003eC. quinoa\u003c/em\u003e plants at 5 DAI in the T4 treatment (60.12 U SOD min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW) when compared to T1 (46.05 U SOD min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In the T1 treatment, SOD activity decreased over time (46.05 at 0 DAI, 43.77 at 3 DAI and 31.18 U SOD min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW at 5 DAI).\u003c/p\u003e \u003cp\u003eHydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) levels in treatments T4 and T6 were lower at 5 DAI, 1030.57 and 1140.21 mmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In contrast, treatments maintained at 25 \u0026ordm;C (T1 to T3) exhibit higher values, above 1652.56 mmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW at 5 DAI. Among treatments involving CoRSV, T3 maintained high H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels, while T6 showed an increase at 3 DAI followed by a decrease at 5 DAI.\u003c/p\u003e \u003cp\u003eAscorbate peroxidase (APX) activity was notably high in T3 at 5 DAI (3.42 nmol ASA min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) correlating with elevated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Conversely, T6 exhibited reduced APX activity (1.71 nmol ASA min⁻\u0026sup1; g⁻\u0026sup1; FW) due to lower H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels. In the T1 treatment, APX activity remained high, exceeding 3.63 nmol ASA min⁻\u0026sup1; g⁻\u0026sup1; FW, indicating an active reduction of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). On the other hand, T4 showed a reduction in APX activity at 5 DAI (0.89 nmol ASA min⁻\u0026sup1; g⁻\u0026sup1; FW).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCatalase (CAT) activity exhibited an increase in the T3 treatment at 3 and 5 DAI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), likely due to the rise in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels caused by virus infection, as similarly observed for APX activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). An increase in CAT activity was also noted in T4, potentially as a response to elevated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels induced by temperature. Overall, CAT levels remained high in \u003cem\u003eC. quinoa\u003c/em\u003e plants, irrespective of abrasive effects or virus presence, ranging from 0.67 nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW in T4 at 0 DAI to 1.39 nmol H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW in T5 at 3 DAI. In T6, CAT levels were consistent, suggesting that APX might be the main contributor to antioxidant activity.\u003c/p\u003e \u003cp\u003eThe quantification of lipid peroxidation, measured via the MDA method, showed variations from 4.68 nmol MDA g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW in T3 at 3 DAI to 18.18 nmol MDA g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e FW in T6 at 3 DAI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among treatments, T6, where plants were inoculated with CoRSV and maintained at 28 \u0026ordm;C, exhibited the highest MDA levels, particularly at 3 and 5 DAI. Treatments maintained at 25 \u0026ordm;C, especially T4 and T6, displayed lower H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and APX activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) but sustained high SOD levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), suggesting superoxide anion as the oxidizing agent.\u003c/p\u003e \u003cp\u003eThe presence of CoRSV was confirmed in T3 and T6 at 10 DAI through electrophoresis gel analysis, with bands observed at the specific size of 708 base pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, variations in the levels of reactive oxygen species (ROS), antioxidant enzymes, and malondialdehyde (MDA) were observed in \u003cem\u003eChenopodium quinoa\u003c/em\u003e plants under different temperature conditions (25\u0026deg;C and 28\u0026deg;C post-inoculation) and treatments with and without inoculation with CoRSV. The ROS pathway in plants begins with the generation of superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e), which is dismutated into hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) by superoxide dismutase (SOD) (Mittler et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Comparing the controls in both environments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), SOD levels decreased in T1, suggesting that a 25\u0026deg;C environment induces less O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e production compared to the 28\u0026deg;C environment (T4). Treatments T1, T2, and T3, maintained at lower temperatures, efficiently converted O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, as indicated by the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Conversely, treatments T4 and T6, exposed to higher temperatures, displayed elevated SOD levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) but continued to exhibit low H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This trend aligns with the observed low levels of APX activity in these treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In treatment T6, plants inoculated with CoRSV and kept at 28\u0026deg;C demonstrated high levels of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, indicating incomplete conversion to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The ambient temperature likely impeded the enzymatic conversion process. Consequently, the elevated O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e levels, compounded by viral infection, may have contributed to increased lipid peroxidation, quantified through malondialdehyde (MDA) levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMDA, a product of lipid peroxidation, is widely regarded as a reliable indicator of cell membrane degradation and a marker of oxidative stress (Abdelkhalek et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, it was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) that \u003cem\u003eC. quinoa\u003c/em\u003e plants inoculated with CoRSV and maintained at a temperature of 28\u0026deg;C (T6) exhibited elevated MDA levels, reflecting significant cell membrane degradation. Previous studies by El-moshaty et al., (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) and Hakmaoui et al., (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) have demonstrated that higher O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e levels lead to increased lipid peroxidation, with subsequent MDA levels declining as tissue structure begins to collapse. The decrease in MDA levels observed in T6 at 5 DAI could be attributed to this structural breakdown, occurring after the peak activity noted at 3 DAI. Several studies corroborate these findings, indicating that the presence of viruses in plants contributes to heightened lipid peroxidation due to increased ROS production (Abdelkhalek et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; P\u0026eacute;rez-Clemente et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sofy et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe damage to the cellular structure of \u003cem\u003eC. quinoa\u003c/em\u003e, as evidenced by the elevated MDA levels in the T6 condition (inoculation with CoRSV and maintained at 28\u0026deg;C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), may play a crucial role in explaining the plant's capacity for long-distance viral movement. Lipid peroxidation damage can compromise the selectivity and stability of cellular membranes, promoting leakage and abnormal interactions (Kar, 2023). This compromised membrane integrity could facilitate the movement of viral proteins and genomes to neighboring cells (Alazem \u0026amp; Burch-Smith, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Excessive O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e can lead to oxidation of the cell wall, making it more fragile and less restrictive to viral movement (Novaković et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This could result in an increase in the size exclusion limit (SEL) of plasmodesmata, as seen in other plant-virus interactions (Zhou et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The combination of damage to the cell wall and plasma membrane may enable the virus to more easily move through cells, shortening the distance to the plant's conducting vessels, where it can initiate systemic infection.\u003c/p\u003e \u003cp\u003eElevated levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in plants, along with other associated changes, induce cell wall fortification and callose deposition (Alazem \u0026amp; Burch-Smith, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Khan et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These processes reduce the SEL of plasmodesmata and hinder viral movement through this pathway (Kumar \u0026amp; Dasgupta, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, high H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels are known to activate systemic acquired resistance (SAR) pathways (Jwa \u0026amp; Hwang, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In \u003cem\u003eC. quinoa\u003c/em\u003e plants maintained at 25\u0026deg;C, the initial oxidative response, coupled with cell wall strengthening due to increased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and the low lipid peroxidation observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), likely restrict viral movement to the local infection site. This suggests that, under these conditions, the host has not suffered significant structural damage that would allow for long-distance viral movement. In contrast, the situation is different for plants inoculated with CoRSV and kept at 28\u0026deg;C, as previously discussed. The higher temperatures seem to impair the plant's ability to mount an effective defense, facilitating viral spread.\u003c/p\u003e \u003cp\u003eFor CoRSV, there are several factors that favor its replication and movement at 28\u0026deg;C. It is suggested that the formation of viroplasm is temperature-dependent, with the higher temperature enabling greater viral content (M. Goodin, unpublished data). This results in increased viral accumulation and the development of systemic symptoms, as observed by Amari, Huang e Heinlein, (2021). Also at high temperature, the movement protein (MP) is more likely to dock at plasmodesma receptor sites or alter its accumulation site, shifting from the replication site to the plasmodesma (Heinlein, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This modification can lead to an increase in SEL of plasmodesmata, facilitating viral movement through adjacent cells until it reaches the conducting vessels. Ultimately, this process leads to the appearance of systemic symptoms, highlighting the temperature's role in promoting CoRSV systemic spread.\u003c/p\u003e \u003cp\u003eIt was observed that CAT levels in the treatment with the virus at 25 \u0026ordm;C (T3) increased at 3 and 5 DAI, whereas in the treatment with CoRSV at 28 \u0026ordm;C (T6), these levels remained somewhat constant, as if CAT had not been activated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Peroxidase levels were more variable (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), with high activity at T3 at 5 DAI. Several factors may explain the behavior of these enzymes: i) the plant may maintain constant or reduced levels of catalase to keep H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels high, thus assigning the dismutation function to APX (Ali et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Riedle-Bauer, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2000\u003c/span\u003e); ii) despite the high rate of CAT, it has a lower affinity for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e than APX, which is why APX increases rapidly during pathogen infection (Mhamdi et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), while CAT increases almost linearly as H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels rise (Mhamdi et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e); iii) the initial damage may have occurred in the chloroplasts, where APX activity is greater, and CAT is predominantly concentrated in peroxisomes (Sousa et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Literature data demonstrate that during viral infection, CAT activity tends to decrease over time, while APX levels increase (P\u0026eacute;rez-Clemente et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Riedle-Bauer, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eC. quinoa\u003c/em\u003e plants infected with CoRSV exhibit the same symptom dynamics as those presented by carnation mottle virus (CarMV), a member of the Tombusviridae family. When kept at mild temperatures (25\u0026deg;C during the day and 18\u0026deg;C at night), only local symptoms appeared. In contrast, when exposed to high temperatures (between 35 and 40\u0026deg;C during the day and 12 to 15\u0026deg;C at night), virus movement within the plant was observed, accompanied by the onset of systemic symptoms. However, Garcı́a-Castillo et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e through immunohistochemical tests, reported that plants kept at both low and high temperatures had the same viral titer, with the pathogen reaching the conducting vessels in both conditions. The authors noted that plant defenses are activated only after the pathogen invades the vascular bundles, and that temperature facilitates a delay in the defense response. Since most plant viruses use the phloem to move throughout the plant, temperature is likely a critical factor in influencing this source-sink relationship (Nagamani et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The growing conditions at 28\u0026deg;C may accelerate the translocation of photoassimilates, and in combination with cellular damage, facilitate the systemic movement of CoRSV. Future studies could explore whether the number of CoRSV copies varies with temperature and host, as observed by Nagamani et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cem\u003eC. quinoa\u003c/em\u003e plants kept in an environment with an average temperature of 28\u0026deg;C for five days after inoculation with CoRSV showed a reduction in hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) accumulation, indicating that the plants are saturated with superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e). This reactive oxygen species can escape from the antioxidant system and lead to lipid peroxidation. Consequently, this damage may affect the cellular structure of the plant, facilitating the systemic movement of the virus. These factors could offer insights into the systemic movement of the virus at 28\u0026deg;C.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they do not have any actual or potential conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e \u003cp\u003eAll authors contributed to the study conception, design, data acquisition, data analysis and interpretation, as well as the drafting of the work. All authors read and approved the fnal manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors would like to thank the Brazilian National Council for Scientific and Technological Development (CNPq), the Coordination of Superior Level Staf Improvement (CAPES) and Foundation for Research Support of the State of Minas Gerais (FAPEMIG) for their support in this project.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdelkhalek, A., El-Gendi, H., Al-Askar, A. A., Maresca, V., Moawad, H., Elsharkawy, M. M., Younes, H. A., \u0026amp; Behiry, S. I. (2022). Enhancing systemic resistance in faba bean (Vicia faba L.) to Bean yellow mosaic virus via soil application and foliar spray of nitrogen-fixing Rhizobium leguminosarum bv. 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Specificity of Plant Rhabdovirus Cell-to-Cell Movement. \u003cem\u003eJournal of Virology\u003c/em\u003e. https://doi.org/10.1128/jvi.00296-19\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Reactive oxygen species, Coffee ringspot virus, Dichorhavirus coffeae, Viral movement, Antioxidant enzymes","lastPublishedDoi":"10.21203/rs.3.rs-5936731/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5936731/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eDichorhavirus coffeae\u003c/em\u003e (CoRSV) can cause local or systemic symptoms in \u003cem\u003eChenopodium quinoa\u003c/em\u003e, depending on the temperature at which the plants are kept after mechanical inoculation. What causes this change in movement is still unknown. Increase in temperature and the presence of viruses can alter reactive oxygen species (ROS), which may be a means of explaining what enables systemic movement. The levels of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), antioxidants enzymes (superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT)), and malondialdehyde (MDA) were investigated in \u003cem\u003eC. quinoa\u003c/em\u003e plants inoculated with CoRSV and kept under two temperature conditions. Six treatments were used: control (T1 and T4), inoculation with phosphate buffer (T2 and T5) and inoculation with CoRSV (T3 and T6). After inoculation, plants from treatments T1, T2, and T3 were kept in a greenhouse at an average temperature of 25\u0026deg;C, and plants from treatments T4, T5, and T6 were kept in a growth chamber at an average temperature of 28\u0026deg;C. Leaf analyses were performed at 0, 3, and 5 days after inoculation (DAI). SOD levels remained high in all treatments. However, plants kept at 25\u0026deg;C showed higher levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and APX activity. Plants with viruses kept at 28\u0026deg;C showed reduced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, APX, and CAT levels at 5 DAI, and high MDA levels. The high amount of MDA present in these plants may indicate that ROS escapes the antioxidant system and causes structural damage in \u003cem\u003eC. quinoa\u003c/em\u003e, thus facilitating the systemic movement of CoRSV.\u003c/p\u003e","manuscriptTitle":"Viral walk: using ROS to explain CoRSV systemicity in Chenopodium quinoa Willd. at high temperature","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-10 08:34:54","doi":"10.21203/rs.3.rs-5936731/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-06-23T04:17:40+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-02-18T10:44:23+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-07T07:55:18+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"European Journal of Plant Pathology","date":"2025-02-07T06:10:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-04T14:48:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Plant Pathology","date":"2025-01-31T08:44:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"935a105c-6972-4736-af5c-fc8afb69aaee","owner":[],"postedDate":"February 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T16:02:35+00:00","versionOfRecord":{"articleIdentity":"rs-5936731","link":"https://doi.org/10.1007/s10658-025-03136-8","journal":{"identity":"european-journal-of-plant-pathology","isVorOnly":false,"title":"European Journal of Plant Pathology"},"publishedOn":"2025-09-26 15:57:22","publishedOnDateReadable":"September 26th, 2025"},"versionCreatedAt":"2025-02-10 08:34:54","video":"","vorDoi":"10.1007/s10658-025-03136-8","vorDoiUrl":"https://doi.org/10.1007/s10658-025-03136-8","workflowStages":[]},"version":"v1","identity":"rs-5936731","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5936731","identity":"rs-5936731","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}