1.Introduction

Herbicides are a class of chemicals that are commonly used to stop, suppress, eliminate, deter, or reduce weed development in crops (Thrall et al., 2011; Vasilescu, Medvedovici, 2005; Zimdahl, 1969). The control of weeds in an economic manner is an important consideration for agriculture and other related industries. Various mechanisms such as absorption, metabolism, translocation, detoxification, and site of action, play a crucial role in the effectiveness of an herbicide (Duke, 1990; Sherwani et al., 2016). The mode of action of herbicides may involve certain pathways leading to up or down regulation of certain enzyme of the weeds or interruption of its normal growth cycle, thus disrupting the regular growth and development of the weed eventually causing its death. Different modes of action of herbicides have been reported namely, lipid biosynthesis inhibitors, amino acid biosynthesis inhibitors, plant growth regulation disruptors, photosynthesis inhibitors, nitrogen-metabolism inhibitors, pigment inhibitors, cell-membrane disruptors, seedling-growth inhibitors (Duke, 1990).

Napropamide (N, N-diethyl-2-(1-naphthalenyloxy) propanamide), trade name Devrinol® (USA, EU, UK, CA) is a selective, systemic amide herbicide. It is absorbed through roots and translocated to the aerial part (Barrett, Ashton, 1981). Napropamide is used in pre-emergence to control many of the annual grass and broadleaf weed species (Toxin and Toxin Target Database (T3DB)).

Napropamide has been reported to inhibit root and shoot growth in corn and tomato, where root growth was inhibited significantly but shoot was affected to a lesser extent (Barret, Ashton, 1981). The reduction in root and shoot biomass has been observed in Brassica napus upon napropamide treatment (Cui et al., 2010). The inhibitory effect of napropamide has also been observed in different wild grasses and plant species like Digitaria sanguinalis, Setaria glauca, and Echinochloa crusgalli (Tseng et al., 1975; Xie et al., 2018; Xie et al., 2019a), Glycine max, Cucumis sativus, Poa annua, and Festuca arundinacea (Qi et al., 2015).

Recent reports also highlight that napropamide is less effective (< 5% control) on perennial weeds such as Cirsium arvense, Trifolium (clovers), Rumex crispus, taraxacum officinale, Solidago canadensis (goldenrods), Elymus repens and Rumex acetosella (Sideman, 2024). The above observation could be attributed to the differences in the root system of these perennials, which often have an extended root system, allowing them to survive harsh weather conditions, and regrow each spring. Napropamide is found to be less effective, as it is known to be effective during preemergence (Dixon, Clay, 2004).

The possible mechanism of napropamide action have been suggested in 1980s. It appears to inhibit root and shoot growth and development, but the underlying mechanism of action is not clearly understood, and hence it has been classified under group 0 by Herbicide Resistance Action Committee (Herbicide Resistance Action Committee, 2024). This study was focused at evaluating the inhibitory effect of napropamide on root growth and development. More than 40% of the commonly used herbicides exhibit enantioselective effects due to their chiral nature (Lin et al., 2008). The enantioselective effect of napropamide has also been investigated in various studies where different enantiomers showed different activities. D (dextrorotatory) (or R (rectus))-enantiomer was found to be more toxic than L (levorotatory) (or S (sinister))-enantiomer in Echinochloa crusgalli (Xie et al., 2018; 2019b), Digitaria sanguinalis, Setaria glauca, and Echinochloa crusgalli (Tseng et al., 1975). D-napropamide was reported to be more toxic than L-enantiomer or racemate to soybean, cucumber, and Poa annua whereas the racemate was more active to Festuca arundinacea (Qi et al., 2015). The commercially available napropamide racemic mixture of enantiomers -D and -L in the ratio 96: 4 was used in our study where -D form is in major proportion which accounts for the major toxic effect.

In tomato plants the inhibitory effect of napropamide on root growth has been reported (Barret, Ashton, 1981, Zilkah et al., 1978). However, these studies have not investigated the role of auxins, reactive oxygen species, and genes regulating auxin transport.

Herbicides may induce oxidative stress by generating reactive oxygen species (ROS) and cause lipid peroxidation in target plants, wherein lipids in the cell membrane gets damaged (Pan et al., 2017) and cause cell death. ROS or its intermediates are formed by reduction-oxidation reactions due to incomplete reduction of oxygen (Sies, Jones, 2020). Recent research highlights the crosstalk between auxins, cytokinins, and ROS. ROS species produced during stress can also impact auxin distribution and response in plant roots (Pasternak et al., 2023).

This research was intended at gaining more insights into the napropamide effect on tomato plant, various biochemical parameters namely total indoles, indole acids (IAA and IBA), protein and nitrogen percentage, ROS and lipid peroxidation and genes regulating the auxins transport were evaluated. Understanding the role of the auxins, genes regulating the auxin flux, oxidative stress and its interplay with auxins in root development would help to decipher the mechanism of action of herbicides in plants.

2.Material and Methods

2.1 Seed germination

Phytotoxicity screening using phytotox system can be used for rapid in vitro screening of any phytotoxic compound (MicroBioTests, 2004). This system complies with ISO 18763: 2016 standards, an international organization adopted standard technique for determining soil quality by evaluating the toxic effects of pollutants on germination and early growth of higher plants (International Standards Organisation, 2016).

It measures the effect of herbicides or toxicants on germination and growth of the young roots after a few days of exposure of seeds to the test compounds. Seeds were germinated using phytotox plates, which allows for direct length measurements of roots and shoots in special transparent test containers, which can be captured digitally. This eliminates the time-consuming measurement efforts inherent to conventional phytotoxicity assays performed in pots.

Seed germination was carried out using phytotox system following the manufacturer’s instructions (Microbiotests, Belgium) as shown stepwise in Figure S1 (A-F) (MicroBioTests, 2004).

A phytotox kit transparent test plate was assembled as follows: a foam pad was placed in the bottom compartment of the plate. On top of the foam sheet a thick white filter paper with 2 mm thickness was placed.

As per the experimental setup, 25 mL of napropamide solutions of different concentrations (0.5 μM to 100 μM) or water as control were taken in syringe and slowly spread over the entire surface of the white filter paper to hydrate it completely. A thin black filter paper was placed on top of the hydrated white filter paper and kept until the black filter was completely wet. Ten seeds of tomato (purchased from the local market) were placed on top of the black filter paper at the specified markings in a row at a distance of one cm between the two seeds. (Figure S1 G). Finally, the cover on the bottom part of the test plate was placed carefully and bulges of the side of the cover were clicked into the corresponding cavities of the bottom part to close the test plate tightly. The plates were incubated in the upright position in the dark at 25 ± 2 °C until germination started. For analysis of root length, shoot length and germination percentage, 3 independent experiments with 10 seeds per concentration of treatment and control were performed.

Seed germination for measurement of different parameters like ROS, Thiobarbituric acid reactive substances (TBARS), Nitrogen content, Auxin levels and gene expression was carried out using plastic mesh basket setup as shown in Figure 1. To obtain sufficient volume of plant material 1 g of seeds were sown per treatment. Three independent experiments were carried out for each analysis.

Performance of this system in our laboratory was evaluated using one of the reference seeds, Sinapis alba (mustard) (purchased from the local market) and its known inhibition by boric acid at 11.5 mM as recommended in phytotox kit (MicroBioTests, 2004). According to the phytotox validity criteria, the mean germination success in the control must be at least 70% and the minimum mean length of the roots must be at least 30 mm. Germination percentage was calculated by the formula given below:

After successful performance evaluation of the phytotox system using mustard seeds as standard, it was further used for analyzing the effect of napropamide on tomato plants. Napropamide used in this study was received as generous gift from UPL, Ankleshwar, India.

Glass trough with diameter of 14.5 cm and height 7.5 cm was filled with 650 mL water as a control or 650 mL of 50 μM napropamide solution. A plastic mesh basket with pore size of 1mm2 and top diameter 15.5 cm, bottom diameter 7.5 cm and height 7 cm was placed on top of the trough to a depth such that its base just touches the water level. A circular hole was pierced in the center of the basket and rolled tissue paper was inserted in the hole to act as a bridge. A circular blotting sheet of diameter 12 cm with a hole in the center (1 cm diameter) was placed in the basket and allowed to become moist. Finally, 1 g of seeds were sprinkled uniformly over the moist blotting sheet and this setup was kept in an enclosed plastic container of dimension 37.5 cm x 24.0 cm in the dark at 25 2 °C for seed germination. Moist filter papers were kept inside the plastic container for humidity.

2.2 Image acquisition and data analysis

Images of phytotox plates for measurement of root and shoot length were captured daily after germination started from 30 cm with a 12 MP Sony IMX 555 1/1.76” sensor camera. Images were captured until root length reached the maximum length of the plate, which occurred at 7–8 days in our experiments. Analysis of germinated seeds involved three parameters, including (1) counting the number of germinated seeds (2) measurement of root length (3) measurement of shoot length. Image analysis program ImageJ was used for subsequent analysis of the images as described in the phytotox manual. A freehand line tool from the ImageJ toolbox was used to measure root and shoot lengths of germinated seeds. The percentage germination of the seeds was calculated. Experiments where more than 70% germination was observed were considered for further analysis.

Percentage inhibition in root and shoot length was calculated using the below formula:

2.3 Measurement of auxin levels

Auxin levels were measured using Salkowski colorimetric method as well as LC-MS/MS as described by Gang et al. (2019). Tomato plants were harvested on day 8 for auxin analysis. Briefly, 1 g of plant material was ground to a fine powder using pestle and mortar in liquid nitrogen. Material was transferred to a 15 mL tube. Five mL of ethyl acetate and 10 μL of 12M HCl were added to the tube and mixed properly by vortexing for 5 min. The sample was centrifuged for 10 min at 4,200 g at 4 °C. Supernatant was transferred completely in glass test tubes and evaporated to dryness in nitrogen evaporator at 40 °C. The residue was redissolved in 5 mL ethyl acetate containing 10 μL of 12M HCl and evaporated again. Finally, the obtained residue was dissolved in 1 mL solvent containing 80% methanol containing and 0.1% formic acid and used for auxin analysis.

Salkowski reagent was prepared as 0.5 M ferric chloride (FeCl3) and 35% perchloric acid (HClO4) in Milli-Q water. For analysis 100 μL sample (as prepared above) was mixed with 100 μL of the Salkowski reagent in a 96-well plate and incubated at 30 °C for 30 mins in dark. One hundred μL of solvent (80% methanol and 0.1% formic acid) was mixed with 100 μL of the Salkowski reagent to serve as blank. Absorbance was measured at 536 nm in a plate reader and compared with a standard indole acetic acid (IAA) curve (10–100 μg mL-1) to determine the unknown concentration (Figure S4).

A liquid chromatography tandem-mass spectrometry (LC-MS/MS) method was used for the determination of IAA and IBA. Sciex Exion LC system coupled with QTRAP5500 MS/MS detector was used for auxin analysis. A binary gradient with mobile phases containing 20% Acetonitrile and 80% of methanol containing 0.1% formic acid in water was used over 8 mins run using Waters X-Bridge C-18 column (3.5 μM, 3x150 mm). Fifty micromolar napropamide treated and control samples (as prepared above) were injected (10 μL in triplicates) in the column maintained at 30°C and autosampler kept at 4 °C. Electrospray ionization (ESI) mode with positive polarity and multiple reaction monitoring (MRM) scan method was used for the quantitation of analytes. The mass to charge ratio (m/z) transitions for analytes IAA and IBA, Entrance potential (EP), Declustering potential (DP), Collision energy (CE) and collision cell exit potential (CXP) in Volts are mentioned in Table S2. The standard curves were plotted with Analyst software (version 1.7.2) of Agilent systems using linear regression with 1/x*x weighting method. IAA and IBA standard curves with linear regression of y = 56,200x-5930, r = 0.9996 and y = 33,800x -2090, r = 0.9997 respectively are shown in Figure S5. Analysis was performed in triplicates for three independent experiments.

2.4 Reactive Oxygen Species (ROS) assay

ROS assay was carried out using Dichlorodihydrofluorescein diacetate (DCFDA) method as per the protocol described by Jambunathan (2010). On day 8, control and 50 μM napropamide treated tomato plants were freshly harvested and ground to a fine powder using liquid nitrogen. One hundred mg of plant powder was taken in a 2 mL tube and 1 mL of 10 mM Tris-HCl, pH 7.2 was added to it. The tube was centrifuged at 12,000 g for 20 min at 4 °C. One hundred μL of supernatant was taken and diluted with 900 μL of 10 mM Tris-HCl, pH 7.2. Ten μL of 1 mM DCFDA was added to the solution followed by vortexing and incubation in the dark for 10 min at room temperature. Plant extract without DCFDA was used as a control to deduct background fluorescence. After 10 min incubation 200 μL of samples were aliquoted in a black 96-well clear bottom plate in triplicates. The fluorescence was measured on a plate reader at an excitation/emission wavelength of 495/522 nm. Analysis was performed in triplicates for three independent experiments.

2.5 Thiobarbituric acid reactive substances (TBARS) lipid peroxidation assay

Lipid peroxidation was estimated by TBARS assay as per the protocol described by Jambunathan (2010). On day 8, freshly harvested tomato plants, control and 50 μM napropamide treated, were ground to a fine powder using liquid nitrogen. Two hundred mg of plant powder was taken in a 15 mL tube, added 4 mL of 0.1% trichloroacetic acid (TCA) and vortexed. The tube was centrifuged at 4000g for 30 min at 4 °C. One mL of supernatant was mixed with 2 mL of 20% TCA and 2 mL of 0.5% thiobarbituric acid (TBA). This mixture was heated at 95 °C for 30 min in a fume hood and cooled on ice before measurement. Aliquot of two hundred μL of samples and blank control were transferred in a transparent 96-well plate. Absorbance was measured at 532 nm and 600 nm on the plate reader. The non-specific absorbance, A600 was subtracted from the values of A532. Malondialdehyde (MDA) concentration was calculated using the linearity equation (y = 0.006 x, with R2 = 0.9996) obtained from MDA standard curve (Figure S2). Analysis was performed in triplicates for three independent experiments.

2.6 Nitrogen and protein content analysis

Nitrogen analysis was carried out using NDA 702 Dumas Nitrogen Analyzer (Velp Scientifica) on a fresh weight basis in tomato plants on day 8. Plant material was ground to a fine powder using liquid nitrogen. Fifty to hundred mg of this powdered material was weighed in a aluminum foil cup and inserted into a nitrogen analyzer for nitrogen content measurement as per the manufacturers protocol. The parameters for nitrogen analysis were kept as oxygen flow rate of 400 mL min-1 and oxygen factor 1.6 mL mg-1. Analysis was performed in triplicates for three independent experiments.

Nitrogen content was calculated based on a standard curve response obtained from oatmeal standard from 5 to 150 mg with known nitrogen percentage of 2.03. Linear regression equation Y = 103.7*X -138.5 and R2 value of 0.9997 was obtained (Figure S3) and used to calculate unknown concentration nitrogen content. Using DumaSoft software (version 3.16.1) expected protein content was derived on mass basis from the percentage of nitrogen obtained.

2.7 Gene expression analysis

To perform gene expression analysis, control and 50 μM napropamide treated tomato plants on day 8, were freshly harvested and ground to a fine powder using liquid nitrogen. 100 mg ground plant material was used for total RNA isolation using TRIZOL method following the manufacturer’s instructions. RNA concentrations were measured using micro-drop plate on a spectrophotometer (Multiskan Sky, Thermo Scientific) at 260 nm. RNA quality was evaluated based on ratio of 260 nm /280 nm absorbance. A 260/280 ratio in the range of 1.8 to 2 was considered ideal for further analysis.

One μg of RNA was reverse transcribed using PrimeScript 1st strand cDNA synthesis kit (Takara) following manufacturer’s instructions with BioRad thermal cycler. Five ng of cDNA was used for qPCR analysis using gene specific primers for acetyl-CoA carboxylase (ACC), Like Auxin Resistant LAX1, PIN-formed (PIN) 2,3,4,7 and Housekeeping gene Ubiquitin ribosomal protein UBI3 (Table S1) and TOPreal qPCR 2X PreMIX (Enzynomics) on a QuantStudio 5 real-time PCR system (Applied Biosystems).

The PCR protocol was as follows: holding stage: 2 min at 50 °C, 10 min at 95 °C; followed by cycling stage: 15 s at 95 °C, 30 s at 60 °C for 40 cycles each. In the final step melt curve analysis was carried out at 60–95 °C with 0.5°C increment at each 0.05s to verify amplification of a single amplicon of UBI3 gene, which was used as an internal control. Raw data obtained from real-time PCR was analyzed using 2 (-Delta Delta C(T)) method (Livak, Schmittgen, 2001). Each PCR reaction was performed in triplicates and three independent experiments were carried out.

2.8 Statistical analysis

For root length inhibition in tomato and mustard seeds using phytotox system, three independent experiments with 10 seeds per treatment as well as for control were evaluated. For evaluating dose dependent effect of Napropramide treatment on root length and shoot length one way ANOVA with Dunnett’s multiple comparisons test was used with GraphPad Prism version 6.

For determination of auxins, ROS, TBARs, nitrogen %, protein content and gene expression analysis in plastic basket setup, three independent experiments were performed. Untreated control and napropamide treated samples were analyzed in triplicates. The difference in control versus napropamide treated samples were analyzed for statistical significance using a two-tailed unpaired t-test with GraphPad Prism version 6.

3.Results and Discussion

For phytotoxicity screening phytotox system was employed, and its performance was evaluated by comparing root length inhibition in mustard seeds by boric acid treatment as recommended in phytotox kit (2004). On day 5, the mean root length was observed to be 71.0± 6.99 mm and 42.7 ± 6.03 mm in control and 11.5 mM boric acid treatment, respectively (Figure S6). The inhibition in root length was found to be statistically significant using unpaired t test with p value of 0.037. Control untreated seeds showed 100 ± 0.0% germination whereas boric acid treated seeds showed 93.3 ± 3.33% germination in three independent experiments with ten seeds each per treatment, fulfilling germination criteria of > 70% as mentioned in phytotox kit. A representative image showing the effect of boric acid on root length in mustard seedlings is shown in Figure S6. The longest root length was reported to be 90.9 ± 6.5 mm and 47.6 ± 7.7 mm in control and boric acid, respectively. Mean inhibition in root length was found to be 39. 6 ± 7.2% upon boric acid exposure and inhibition in the longest root was 48.2 ± 5.8%. Inhibition % values for both parameters were within acceptable limits as mentioned in phytotox validity criteria i.e. mean % inhibition of the mean root length was ranging between 22–65% and mean % inhibition of the mean length of the longest root was between 28–60% This validation criteria are based on the ring trial data obtained from 28 different laboratories (Baudo, 2011). Our results with root length inhibition of mustard seeds are also in line with these observations.

At napropamide concentrations ranging from 0.5 μM to 100 μM tomato seeds showed hindered root growth. Images of root length were taken from day 5 to day 8. A representative analysis of root length inhibition on day 6 has been given in Table 1. An inhibition of 22.3 ± 3.0% to 75.1 ± 1.2% was observed in root length, where it varied from 20.0 ± 1.1 mm to 62.5 ± 8.0 mm at different tested concentrations and 80.4 ± 3.1 mm for control in three independent experiments with 10 seeds per concentration on day 6 (Figure 2).

Napropamide showed a dose-dependent inhibition in root length whereas no significant effect was observed on shoot length on day 6 (Table 1, Figure 2). The inhibition in root length was found to be statistically significant based on unpaired t test with p value of 0.0001 to 0.037. This inhibition in root length was found to be linear in the concentration range of 5 μM to 50 μM of napropamide, with linear regression equation of y = 0.6262x + 42.019 and R² value of 0.9856 (data not shown).

A similar concentration dependent inhibition was also observed in concentration range of 5 to 50 μM for day 5, 7 and 8 with R² value of 0.9277, 0.9138 and 0.8869 respectively. Hence, the two napropamide concentrations of 5 μM and 50 μM were selected for further experiments. A representative image of napropamide induced root length inhibition on day 8 has been shown in Figure 3. In three independent experiments at 5 and 50 μM with 10 seeds each, the mean root length ranged from 44.9 ± 6.4 mm to 78.1 ± 2.9 mm for untreated control, 34.1 ± 3.7 mm to 50.3 ± 9.5 mm for 5 μM napropamide and 21.7 ± 1.1 mm to 25.1 ± 1.8 mm for 50 μM napropamide, respectively from day 5 to day 8 of seeding (Figure 4). The differences in inhibition were found to be statistically significant using one way ANOVA with Dunnett’s multiple comparisons test.

The percentage inhibition in mean root length upon napropamide exposure at 5 μM and 50 μM for day 5 to day 8 have been shown in Table 2. The seed germination percentage was also calculated based on the total number of seeds germinated out of 10 seeds planted in each setup. In The mean germination percentage was 73.3 ± 3.3%, 77.0 ± 8.8% and 90.0 ± 5.8% for untreated control, 5 μM napropamide and 50 μM napropamide, respectively.

The inhibition in shoot length was measured at day 8 and found to be statistically insignificant with t-test; 17.2 ± 8.3% at 5 μM and 16.5 ± 14.1% at 50 μM with average shoot length of 52.2 ± 3.9 mm, 42.7 ± 2.9 and 42.8 ± 5.3 in control, 5 μM and 50 μM napropamide, respectively (Figure 5). The evaluation of morphological parameters clearly indicated that napropamide was affecting the plant root system while shoot development is affected to a lesser extent. Our results are comparable to the findings in other reports on different plant species like tomato, corn and rapeseed where inhibition has been observed in the similar range as in our findings i.e. 46% and 69% at 5 μM and 50 μM, respectively.

Napropamide has been reported to inhibit root growth significantly in corn and tomato seedlings, however shoot was affected to lesser extent (Barret, Ashton, 1981). In tomato root length was found to be inhibited by 40% and 82% and shoot length by 3% and 39% on day 5 at 0.1 μM and 10 μM napropamide treatment respectively. Similar treatment with napropamide in corn seedlings resulted in an inhibition of 1% and 65% in roots and -3% and 33% in shoots. Zilkah et al. (1978) has also demonstrated inhibitory effect of napropamide in tomato plants. The root and shoot biomass in Brassica napus (rapeseed) seedlings have also been shown to reduce by 46% and 28% respectively upon exposure to napropamide for 5 days at 8 mg/L (~ 30 μM) (Cui et al., 2010).

Auxins are involved in abiotic stress adaptation in plants (Bielach et al., 2017 ; Simon et al., 2011). The prominently occurring endogenous auxins contains indole backbone, which can be quantified using colorimetric Salkowski method.

Indole acid content in 50 μM napropamide treated tomato plants was found to be 13.2 ± 0.9 micro g mL-1 against 10.2 ± 0.9 micro g mL-1 in control on day 8 in three independent experiments (Table 3). Total indole acids were observed to be higher in napropamide treatment statistically significant using unpaired t test with p = 0.0151. The colorimetric method quantifies various types of indole compounds produced as a product of tryptophan metabolism (Gang et al., 2019). However, the colorimetric method doesn’t discriminate between IAA and IBA.

Hence, specific indole acids, IAA and IBA were further quantified by LC-MS/MS for better clarity. IAA content in control and napropamide treated plants was found to be 31.3 ± 6.1 ng mL-1 and 88.8 ± 16.2 ng mL-1 (2.8-fold) based on LC-MS/MS analysis (Table 3) which was found to be statistically significant using unpaired t test with p= 0.0045. IAA level was higher in napropamide treated plants as compared to untreated plants. Similarly, a higher IBA level of 55.2 ± 3.4 ng mL-1 (2-fold) was observed in napropamide treated plants in comparison to the control which was 27.7 ± 2.5 ng mL-1 which was also found to be statistically significant using unpaired t test with p = 0.0004 (Table 3). A representative image of LC-MS analysis for control and treated samples for IAA and IBA is shown in Figure 6. Quantification by both the methods showed a similar trend of higher indole content in napropamide treated samples where, 1.3-, 2.8- and 2.0-folds higher content was observed in total indole compounds, IAA and IBA respectively.

Auxins play a cardinal role in multiple aspects of plant growth and development, primarily the root system in coordination with other phytohormones (Qin et al., 2019). Auxins are predominantly synthesized in shoot apical meristem and leaf primordia and subsequently transported to the roots.

The outcome of our results, despite the higher concentration of auxins in napropamide treated plants, the root development is compromised as observed in morphological analysis. The biphasic response of these hormones has been well-reported in various studies. Auxins in optimum levels are reported to enhance plant growth and development, however, a higher concentration of auxins may be inhibitory for the plant. The response of different plant tissues to auxin has been reported to be concentration dependent. Auxins at higher concentrations tend to be inhibitory, therefore optimal endogenous auxin levels must be firmly regulated (Zhang et al., 2022). Multiple mechanisms have been reported for regulating auxin homeostasis, which includes dynamic biosynthesis and transport, degradation, and formation of conjugated IAA (Simon et al., 2011, Zhang et al., 2009). Conjugation of auxin with amides or sugars is one of the important regulatory mechanisms to control auxin activity (Ludwig-Muller, 2011). Treatment of plants with exogenous IAA conjugated with Tryptophan has been shown to suppress the root responses to the active endogenous IAA suggesting negative control of auxin activity via auxin conjugates (Staswick, 2009).

In ROS assay using DCFDA method, the relative fluorescence units (RFU) value for napropamide treated (50 μM) tomato plants at day 8 was observed to be 75887 ± 3958 in comparison to control which was 64619 ± 7875 (Table 4). In three independent experiments with three replicates, a marginal increase of 1.23 ± 0.23-fold ROS level was observed upon napropamide treatment indicating oxidative damage at the cellular level, which might be affecting the plant growth. Increased ROS production may lead to metabolic disorder and oxidative destruction of the cells, primarily by oxidative degradation of lipids in turn affecting cell viability and function. Most of the disturbances caused by herbicide treatment are linked to ROS generation and subsequent oxidative stress (Caverzan et al., 2019).

TBARS assay is the most widely used method for determining lipid peroxidation (Yagi, 1998). MDA is produced during the process of lipid peroxidation by the decomposition of polyunsaturated fatty acids which is often used as a marker for lipid peroxidation in studies related to oxidative stress and redox signaling in plants (Wang et al., 2016). MDA further reacts with thiobarbituric acid and quantified colorimetrically. The MDA concentration was found to be 3.2 ± 0.5 μM in napropamide treated (50 μM) tomato plants as compared to 2.3 ± 0.1 μM in control (Table 4), which is 1.4 ± 0.17 folds higher and found to be statistically significant by unpaired t test (p = 0.0378). Three independent experiments were performed with 3 replicates each. Increased levels of MDA have also been observed in other plant species in response to napropamide treatment. Echinochloa crusgalli showed an increase of 2.33- and 2.28-fold MDA in roots and shoots respectively in response to micromolar concentrations of napropamide (Xie et al., 2019a). Increased lipid peroxidation in response to napropamide has also been reported in Brassica napus with 2.0 and 2.4-fold higher TBARS in roots and shoots, respectively (Cui et al., 2010). The marginal rise in ROS and MDA indicates that oxidative stress could also be a contributor to the induction of stress in the napropamide treated plants.

Herbicides treatments have been reported to affect the nitrogen and protein content on legume plants in a dose-specific manner (Zaidi et al., 2005, Khan et al., 2004). In three independent experiments with three replicates at day 8, both 50 μM napropamide treated as well as control samples showed 0.57 ± 0.02% and 0.49 ± 0.03% nitrogen levels respectively (Figure 7). Similarly, percentage of protein was also found to be 3.4 ± 0.24% and 3.2 ± 0.07% in control and treated samples respectively (Figure 7). This establishes that napropamide did not affect nitrogen and protein content significantly. Our experimental setup was designed to study the short-term effects of herbicides up to 8–10 days. For long term studies on plants soil or appropriate growth media is essential. In our experiments, marginal change in nitrogen and protein content may be due to the shorter time window for evaluation and or this could also be due to lower tested concentration.

The pattern of auxin distribution within the plant is achieved through a complex and well-coordinated transport system known as polar auxin transport (Friml, 2003) which is a key factor for plant growth and development of different organs and its reaction to its environment. Herbicide stress may result in altered expression of auxin transporters like LAX1 and PINs, which in turn can affect root growth. Increased PIN expression may modulate auxin signaling pathways, aiding plant survival during stress. (Liu et al., 2014). Therefore, expression analysis of auxin transporter genes was carried out to study the effect of napropamide at the molecular level. AUX1/LAX1, four PIN-FORMED (PIN2, PIN3, PIN4, PIN7) and acetyl-CoA carboxylase (ACC) were selected for mRNA expression analysis.

It has been reported that napropamide may also inhibit lipid/fatty acid biosynthesis by inhibiting ACC enzyme activity which catalyzes the first step in fatty-acid synthesis (Yang et al., 2010). AUX1/LAX1 family genes are the major auxin influx carriers and members of the PIN family are major efflux carriers (Swarup, Bhosale, 2019). In gene expression analysis, all 6 genes showed above 1.5-fold upregulation in response to napropamide treatment (Figure 8). PIN3 showed the highest upregulation of 3.6 folds. Other genes, PIN4, PIN2, and LAX1 showed an increase of 2.7, 2.4 and 2.1 folds respectively. ACC and PIN7 were the least upregulated genes with an increase of 1.9 folds. Upregulation in LAX1, PIN3 and 4 were found to be statistically significant based on unpaired t-test. These differences could also be contributed to the consequences of the herbicide effect. It has been reported that spatio-temporal expression patterns of PIN formed (PINs) auxin efflux carriers and subcellular localization synergistically modulate stress-induced reorientation of growth (Bielach et al., 2017).

Auxins are synthesized in shoots and transported downwards to the roots either through bulk flow in the phloem or through polar auxin transport. Higher polar transport of auxins inside the cells by influx transporter (LAX1) is expected to cause root elongation. But in our case, as most of the efflux transporters (PINs) are highly upregulated in comparison to influx transporters, the overall auxin efflux rate is higher which might be causing a reduction in root length. These results indicate the expression status of transporters in combined plant samples (root, shoot, stem). Expression analysis of these transporters in individual tissues could give an exact idea about the auxin transportation. Guan et al. (2019) have studied the expression of these transporters in different tissues of tomato (root, shoot, leaf). The expression of LAX1 and most of the PINs was highest in leaves followed by shoots and roots. Detailed spatio-temporal gene expression study might give a clear understanding of the effect of napropamide on auxin transport system. This would be helpful in deciphering the correlation between higher auxin content and root length inhibition upon napropamide treatment.

In this study, key parameters were evaluated namely, morphological analysis – root and shoot length, biochemical analysis – estimation of indole acids (IAA and IBA), proteins, nitrogen, measurement of ROS, lipid peroxidation and gene expression to determine the inhibitory effect of napropamide.

Significant differences in root length and in biochemical analysis upon napropamide exposure have been observed. Auxin-related genes have also shown upregulation in response to napropamide treatment. It can be concluded that napropamide could potentially perturb one or more of the regulatory mechanisms involved in auxin homeostasis such as dynamic biosynthesis and transport, degradation, and formation of conjugated forms of auxin. This may result in higher accumulation of auxins in napropamide treated plants thereby causing the inhibition of root development and elongation. Therefore, a detailed dissection of the auxin regulation machinery is required to get a clearer picture of the napropamide effect. A holistic approach at the molecular level using modern OMICS tools like NGS, proteomics, and metabolomics can be employed.

4.Conclusions

This study demonstrates the inhibitory effect of napropamide on tomato plants, by evaluating various morphological and physiological parameters namely root length inhibition, seed germination, perturbations in auxin levels in shoot and root, ROS and lipid peroxidation as well as evaluation of gene expression of key auxin related genes. These results provide a simple, rapid and user-friendly method for in vitro screening of herbicides involving cultivation, analysis of the relevant biomarkers and corroboration with gene expression, for screening of new herbicides and their effects on weeds.

Acknowledgements

Authors kindly acknowledge Dr. Vilas Sinkar for his guidance and Dr. Nadeem Khan for his input in the manuscript.

References

Edited by

Publication Dates

History