Trade-offs in Root and Shoot Growth in Forage Pea [Pisum sativum (L.) arvense] with Foliar Applications of Synthetic Elicitor DPMP (2,4-Dichloro-6-{(E)-[(3-Methoxyphenyl) Imino] Methyl} Phenol) and SA (Salicylic Acid)

Plant species are naturally evolved to cope with environmental fluctuations. However, climate change and excessive abiotic and biotic stress pressure reduce plants’ ability to be productive enough for the increasing population (Snowdon et al., 2021). Even though there are substantial developments on the resistance/tolerance to abiotic and biotic stresses, there is still a large gap between genotypic potential and current crop production levels (Jaggard et al., 2010). Drought and salt stress as two of the major limitations of agricultural production are becoming a growing threat worldwide, affecting an extending portion of the agricultural land (Yang and Guo, 2018). There have been a significant number of studies to reduce or eliminate the negative effects of osmotic stresses on plants (Nagel et al., 2014; Robin et al., 2021; Kouki et al., 2021), however, the success rate is quite limited due to the complex nature of the abiotic stresses and plants response mechanism (Witcombe et al., 2008; Tardieu and Tuberosa, 2010). Abiotic stresses such as ionic and osmotic stresses generally reduce or completely prevent photosynthesis and limit growth and production (Liang et al., 2018).
       
The plant defense system has a complex regulatory mechanism protecting plants against diseases (Tsuda et al., 2008; Sato et al., 2010). Salicylic acid (SA) is a phytohormone and one of the most important regulatory components of plant defense systems. Numerous reports showed the power of exogenous SA application to induce the plant defense system against a variety of pathogens (White, 1979; Bektas and Eulgem, 2015). Subsequent research demonstrated the activity of SA as a plant growth regulator. It enhances plant adaptation not only to biotic stresses but also against abiotic stresses such as drought, heavy metal and cold (Wani et al., 2016; Zhao et al., 2017). Moreover, increasing evidence has shown improved plant tolerance against drought by exogenous application of SA (Samota et al., 2017). Plant defense elicitors, aka ‘Plant activators’ are inducers of the plant defense system and protect plants against a variety of pathogens (Reddy, 2013; Bektas and Eulgem, 2015). Many studies have shown that in addition to SA, other characterized plant defense elicitors also increase plant adaptation to abiotic stresses. g-aminobutyric acid (GABA) and b-aminobutyric acid (BABA), Acibenzolar S methyl (ASM), Sodium silicate and Saccharin were reported to enhance drought tolerance in broccoli (Jespersen, 2017; Venegas-Molina et al., 2020). Previous reports demonstrated that in addition to biotic stresses, plant defense elicitors may have the potential to increase abiotic stress tolerance and reduce the severity of stress factors including drought stress. 2, 4-dichloro-6-{(E)-[(3-methoxyphenyl) imino] methyl} phenol (DPMP) is a novel synthetic plant defense elicitor that has been shown to induce defense responses in Arabidopsis thaliana and tomato (Bektas et al., 2016) but its activity on abiotic stress tolerance has not been evaluated.
       
Forage plants are mostly legume or Poaceae species, that are mainly grown for fresh biomass and dry herbage production. Their sustainable production is the key to continuous farm animal and dairy production (Martin et al., 2017). Legume forage species are the most preferred forage group, due to their high protein content and nitrogen fixation advantage compared to Poaceae species (Chen et al., 2018). Forage pea [Pisum sativum (L.) arvense] is a legume species that is mostly used for fresh or dry herbage animal feeding (Çaçan et al., 2019). It is commonly grown around the world and is considered to be sensitive to salt stress (Grozeva et al., 2019). Improving abiotic and biotic stress tolerance levels of the current cultivars, or breeding new cultivars with better stress tolerance are considered to be the main paths to a climate-resilient production of forage pea. There have been several reports of forage pea seedling growth (Demirkol et al., 2019; Acikbas et al., 2021a), but none of the previous studies evaluated the effect of plant defense elicitors, such as SA and DPMP on osmotic stress tolerance and their role on growth in forage pea. Therefore, this study aimed to evaluate the effects of SA and DPMP foliar application on the osmotic stress tolerance, root-shoot growth and seedling vigor of forage pea, under controlled conditions.

Forage pea cultivar GAP Pembesi is selected as the model plant for chemical application × osmotic stress tolerance interactions due to its known sensitivity to salt stress (Tekeli and Ateş, 2011) and known root developmental properties (Acikbas et al., 2021b). The study was conducted under controlled conditions in the Department of Agricultural Biotechnology, Siirt University, Siirt, Turkey (37^o58'13.20"N -41^o50'43.80"E). Mean temperature and relative humidity ranged between 25-27oC and 60-70%, respectively. The experiment was conducted under daylight conditions at 12:12h day/night. The study was established according to completely randomized design with three replications and ten plants per replication. Polyethylene glycol (PEG 8000) is applied as 10% and control treatment had no PEG treatment (0%).
       
SA was ordered from Sigma-Aldrich Chemie GmbH, Germany. DPMP was kindly obtained from Prof. Dr. Thomas Eulgem, University of California, Riverside, USA. SA and DPMP dissolved in 100% DMSO until 50 mM and 5 mM stock concentrations, respectively and then diluted with distilled sterile water to indicated concentrations. 100 μM SA, 10 μM DPMP, or control solution (0.2% DMSO) were applied as a foliar spray at the 7^th and 10^th days of growth. The growth system is a semi-hydroponic version of the modified cigar roll technique (Zhu et al., 2005; Acikbas et al., 2021a).
       
Seed surface sterilization was made with 70% ethyl alcohol (C₂H₅OH) and 5% sodium hypochlorite (NACIO) for 5 minutes and rinsed under running water for one minute. Seeds of similar size were placed between germination papers (60×40 cm) as ten seeds per germination paper, the method is slightly modified from (Hohn and Bektas, 2020). Each set of germination paper is covered with a second layer and rolled to fit in cylindrical containers filled with the specified solutions, 10% PEG 8000, or distilled water. The experiment was conducted on May 20^th, 2021 and completed on June 7^th, 2021.
       
At the end of the experiment, each roll is separated and images of each plant are taken with a handheld scanner (Iscan Color Mini Portable Scanner) at 300 DPI resolution. Image analysis was performed manually using ImageJ (Rueden et al., 2017) software. Root and shoot growth (Acikbas et al., 2021a) and root architecture traits (Merrill et al., 2002; Acikbas et al., 2021a) given in Table 1 were evaluated with image analysis. Stress tolerance-related calculations were made according to Moursi et al., (2020).
 

       
The effects of SA and DPMP applications on osmotic stress tolerance and seedling growth vigor were analyzed with analysis of variance (ANOVA) using Statistix 10 software (Analytical Software; Tallahassee, FL, USA). Variance groupings were made using the Least Significant Difference (LSD) multiple comparison test (Steel et al., 1997).

Root-shoot growth and seedling vigor
 
Shoot length (SL) under no stress conditions was similar between SA, DPMP and control. The SL under osmotic stress was also similar within the PEG treated group (Fig 1A). TapRL was the longest under SA and control compared to DPMP, while the control had better TapRL under osmotic stress. Similar results were also seen in TotalLatRL and TotalRL (Fig 1C and D). DPMP significantly improved aLatRL under untreated conditions, but it did not show the same effect under stressed conditions. NOLatR was better in control-treated and untreated conditions. SFW, SDW, RDW, SFW/RDW and SDW/RDW were similar within each group. On the other hand, RFW was higher in SA-untreated and control-untreated conditions. These results overall suggest that most of the root traits were similar within SA, DPMP and control untreated conditions and within treated conditions. We can suggest that SA and DPMP, neither inhibited nor enhanced some seedling root traits, except aLatRL, NOLatR, RFW and SFW/RFW ratio (Fig 1). Previous reports (Larqué-Saavedra and Martin-Mex, 2007) suggested growth-enhancing effects of SA can be seen under low SA doses. The dose we applied here was the spray dose used for biotic stress factors. So, the optimal SA dose for growth-enhancing effect and biotic stress tolerance may be different. Koo et al., (2020) report significant biotic stress eliciting effects for SA, while results for plant growth under stress and optimum conditions were dose-dependent. SA becomes an inhibitory agent at high doses. The current dose we applied may be high for the growth enhancement of some root traits in forage pea. There is a need for the evaluation of different doses to define the right dose for growth enhancement in forage pea and other crops. These factors, tend to enhance plants’ response to biotic stresses, but their effect on plant growth and vigor was not clear for some traits.
 

 
SA had higher values compared to DPMP and control on SL, TotalRL and SFW untreated and SFW/RFW treated conditions. DPMP had higher values in aLatRL, RDW and SDW/RDW untreated conditions (Table S1). However, some of these differences were not statistically significant (Fig 1). Pearson’s correlation coefficients showed significant positive correlations between SL, SFW, RFW, SDW and RDW. TotalLatRL, aLatRL and TotalRL were in a positive correlation with shoot and root biomass traits (Table 2). Correlation analysis confirmed the above and below-ground growth interactions (Bektas et al., 2020). Above and below ground growth and vigor supports each other under normal and moderate stressed conditions, while under severe or long-term stress root-shoot relations may change based on genotypic potential and genotype × environment interactions (Ye et al., 2018).
 

 

 
Stress tolerance
 
The effects of SA and DPMP on plant growth and stress tolerance were evaluated under PEG-8000 treated and untreated conditions. To clearly see the effect of each treatment, a total of six different combinations were applied; SA, DPMP and control with and without osmotic stress. Therefore, we were able to compare the role of each elicitor under normal growth and stressed conditions. Reduction on TotalRL, RFW and RDW was the minimum on control, while reduction rates on SFW and SDW were similar in DPMP and control. Control untreated had the highest drought tolerance index (DTI) values compared to control-treated applications. It was followed by DPMP, which was close to control and SA had the least DTI values (Table 3). These results suggest that, even though SA and DPMP are common biotic stress response elicitors, they may not enhance plant growth under abiotic stress if the optimum dose is not applied (Hayat et al., 2010; Koo et al., 2020). Hayat et al., (2010) and references therein reported enhanced root and shoot growth at low doses of SA. Also, DPMP improved root length at lower concentrations while higher doses reduced root length on Arabidopsis thaliana (Bektas et al., 2016). The effect of this novel plant activator, DPMP, on plant growth and stress tolerance needs to be determined for defining appropriate doses for DPMP at foliar application.
 

This study aimed to evaluate the effects of two different biotic stress tolerance elicitors on seedling growth and development under osmotic stress and non-stressed conditions. It was seen that for some traits SA and DPMP did not inhibit or enhance plant growth compared to control under treated and untreated conditions, while their effects on aLatRL were noteworthy. Under untreated conditions plants sprayed with DPMP had significantly higher (5.57 cm plant^-1 in DPMP, 4.53 cm plant^-1in SA and 3.01 cm plant^-1 in control) aLatRL values. This was possibly due to inhibited NOLatR compared to control. Control had much higher (14.59 plant^-1) NOLatR than SA (9.89 plant^-1) and DPMP (6.97 plant^-1). Based on our results, these two defense elicitors can be applied as a foliar spray to enhance plant immune responses against pests and diseases, without any issues on growth limitation.

None.