Redox regulation of adventitious root formation through downstream changes in hormonal system in mung bean [Vigna radiata (L.) R. Wilczek]

ROS acts downstream of many signaling pathway initiated by plant growth hormones leading to the genesis of adventitious root formation [1-6]. ROS are generated in response to any physical damage or wound [7], acting as a second messenger in response [8,9]. Removal of primary roots may induce a wound signal including ROS that intiates ARF for water and nutrient uptake from the surroundings [3,4,10-13] Removal of primary roots increased the endogenous H2O2 in water treated cucumber explants in compared to the DPI (Diphenyleneiodonium, an inhibitor of NADPH-oxidase) treated explants of cucumber where the primary root removal increased the endogenous H2O2 but it got lowered after few hours and remained at lower concentration unlike in control treatment where it reached its higher peak after 3 hr then declined to its lowest concentration after 11 h, increasing again attaining second peak after 17 h. exogenous application of 20–40 mM H2O2 or 10 mM Indole Acetic acid (IAA) signifi cantly increased the number of AR in cucumber seedling explants and 10–50 mM H2O2 signifi cantly increased the fresh weight of adventitious roots. 2 mM ascorbic acid, 100 U CAT (Catalase) or 1 mM DPI eliminated the root promoting effect and the effects of IAA were eliminated by 4 mM ascorbic acid, 100 U CAT or 5 mM DPI which were partly rescued by exogenous H2O2 clearly indicating the role of H2O2 in ARF [14]. Recent study in cucumber, ROS, auxin and ethylene accumulated during water logged condition. Auxin and ethylene both induced the ROS generation. Both ethylene and auxin Abstract


Introduction
ROS acts downstream of many signaling pathway initiated by plant growth hormones leading to the genesis of adventitious root formation [1][2][3][4][5][6]. ROS are generated in response to any physical damage or wound [7], acting as a second messenger in response [8,9]. Removal of primary roots may induce a wound signal including ROS that intiates ARF for water and nutrient uptake from the surroundings [3,4,[10][11][12][13] Removal of primary roots increased the endogenous H 2 O 2 in water treated cucumber explants in compared to the DPI (Diphenyleneiodonium, an inhibitor of NADPH-oxidase) treated explants of cucumber where the primary root removal increased the endogenous H 2 O 2 but it got lowered after few hours and remained at lower concentration unlike in control treatment where it reached its higher peak after 3 hr then declined to its lowest concentration after 11 h, increasing again attaining second peak after 17 h. exogenous application of 20-40 mM H 2 O 2 or 10 mM Indole Acetic acid (IAA) signifi cantly increased the number of AR in cucumber seedling explants and 10-50 mM H 2 O 2 signifi cantly increased the fresh weight of adventitious roots. 2 mM ascorbic acid, 100 U CAT (Catalase) or 1 mM DPI eliminated the root promoting effect and the effects of IAA were eliminated by 4 mM ascorbic acid, 100 U CAT or 5 mM DPI which were partly rescued by exogenous H 2 O 2 clearly indicating the role of H 2 O 2 in ARF [14]. Recent study in cucumber, ROS, auxin and ethylene accumulated during water logged condition. Auxin and ethylene both induced the ROS generation. Both ethylene and auxin induced AR were inhibited by NADPH oxidase inhibitor, DPI, suggesting a signifi cant role of ROS in AR morphogenesis [15]. The Plant growth hormones like auxin, ethylene, NO etc. make use of ROS and other PGRs like Gibberelic acid, Abscisic acid, Jasmonic acid (GA, ABA and JA) etc. in initiating ARF. ABA, GA and JA play an important role in plant responses to biotic and abiotic stress. There is less study in the direct cross talk of ROS with ABA, GA and JA during ARF.
SA, an endogenous plant growth regulator which is found to have both stimulatory and inhibitory effects on ARF [16][17][18][19][20][21]. It works synergistically with auxin in IAA-induced ARF [22]. In a recent work, it has been shown that SA induces ARF in cucumber through competitive inhibition of the IAA-Asp synthetase activity of CsGH3.5 (Gretchen Hagen3.5) leading to the increase in free IAA content. Application of 50 μM SA could lead to activation of cyclin and CDK gene expression indicating its effect on adventitious rooting during induction phase [23]. Unlike methyljasmonate, SA increased the scopolamine in adventitious root cultures of Scopolia parvifl ora but did not show any negative effect on root growth, infact SA exposure for relative short time and during active growth phase, all the concentation studied slightly increased the root growth index and did not stimulate root browning [24]. In a study of Tomato stem rooting, it was found that in addition to auxin, ABA, zeatin and SA may play a complementary role in the induction, initiation and emergence of the developing AR and hormone homeostais is important during all the stages of ARF [25]. However, some workers have found that SA inhibited adventitious root formation. It decreased the weights of roots in mung bean hypocotyls cuttings [20]. It is reported to promote IAA decarboxylation and reduces rooting when applied duing the intial days when auxin enhances rooting. During the later phase, when auxin inhibits rooting, it promotes outgrowth of root primordia [21].
ROS supposed to act downstream of SA and application of ROS scavenger DMTU and NADPH Oxidase inhibitor DPI both led to the reduction of ARF in Mung bean hypocotyl [5,16]. Mutants in the alleles eds5-1 and eds5-2 (SA induction defi cient mutant), unable to synthesize SA produces signifi cantly lower number of ARF as compared to the wild type [16]. In most of the cases, SA found to induce ARF via up-regulation of H 2 O 2 formation [16] indicating the implication of ROS [16,18]. Salicylic acid increases the production of ROS via changing the antioxidative defnce system like inhibiting the activity of Catalse enzymes and increasing the activity of H 2 O 2 formation enzymes [16,26].
GA, a tetracyclic di-terpenoid compound, is a plant hormone involved in plant growth and development. It stimulates Dormancy, seed germination, fl owering and sex expression, embryo development, trigger transitions from meristem to shoot growth, juvenile to adult leaf stage, grain development and also interacts with different environmental conditions like Light, temperature etc [29][30][31][32][33][34]. GA also has a stimulatory effect on root formation [35][36][37]. Gibberellic acid at low concentration (10 -9 -10 -6 M) promotes rooting, might because of mobilization of low-molecular weight carbohydrates from shoot to lower part. In low irradiance, light may modify the rooting response of GA 3 treated cuttings of stock plants [38] . Plant cuttings of Pisum sativum formed higher number of roots at an irradiance of 16 W m -e but lower rooting in higher irradiance then this [39]. GA 3 dissolved in water promoted rooting signifi cantly at 10 -7 M and 10 -8 M [40]. It is also suggested that root initiation required an appropriate combination of GA 3 and auxin [41]. Higher GA 3 or the lower IAA concentration resulted in more roots in Persicaria perfoliata [42]. GA 3 increased the number of roots and the effect increased with concentration (from 10 and with 100 mg/L) in stem cuttings of Ipomoea fi stulosa [39].
However many work have also reported that GA 3 inhibits ARF [39,[43][44][45][46][47]. Gibberellic acid in high concentration inhibited root formation [39] and may probably inhibits the outgrowth of root primordial [40]. In some cases, GA treatment reduced the cell division in established primorida and also blocks the action of IAA in some process subsequent to the primordial initiation [48]. This inhibition might be due to a competitive diversion of mobilized reserve food materials towards the shoot than to the root meristems [47]. GA-treated barley (Hordeum vulgare) aleurone layers causes rapid death of all cells when incubated with H 2 O 2 but ABA-treated did not cause cell death upon incubation with same. Also there was decrease in the amounts and activities of ROS scavenging enzymes, like catalase (CAT), Ascorbate Peroxidase (APX), and superoxide dismutase (SOD) in aleurone layers treated with GA. on the other hand, the amount and activity of CAT and Cat2 mRNA was found to be increased in the ABA-treated layers which also maintains high amounts of ascorbate peroxidase and superoxide dismutase, whereas GA causes rapid reduction in the amounts of these enzymes resulting in oxidative stress leading to cell death [49]. Also, it has been reported that GA-treated aleurone protoplasts are less able to tolerate endogenous or exogenously applied H 2 O 2 than ABA-treated protoplasts [50]. These fi ndings suggest that GA 3 might suppress the antioxidative defence system resulting in oxidative stress and may promote ARF at low concentration by encouraging the ROS formation with downregulating effect on ROS scavenging enzymes. Gibberellins also have a negative effect on AR formation in poplar. Here the mutants gibberellins insensitive (gai) and repressor of GA1-like1 (rgl1), affected in gibberellins perception and responses to gibberellins as well, AR formation remains unaffected [51].
ABA, also called stress hormone, is an isoprenoid phytohormone [52]. It regulates various physiological roles such as stomatal closer, leaf senescence, Bud dormancy, seed germination, osmotic regulation, cuticular wax accumulation, modulation of root architecture and also it controls various downstream processes related to abiotic and biotic stresses [53,54]. Li, et al. [55] have showed that ABA promotes adventitious rooting in mung bean by reducing the activities of antioxidative enzymes (CAT, APX) and antioxidant levels (GSH, AsA) similar to the work of Fath, et al. [49]. ABA also acts as a second messenger in the signaling pathway of adventitious rooting. ABA is found to up-regulate CsHO1 cucumber Heme Oxygenase (HO) gene that forms carbon monoxide that helps in the adventitious rooting [56]. Endogenous H 2 O 2 was suggested to be involved in ABA-induced adventitious root development under drought stress in cucumber [57]. Later on comparative proteomic analysis of key proteins have revealed Citation: Kora [58]. ABA at a concentration of 5 x 10 -5 M promoted adventitious root development, and the root number increased when it is applied along with IAA [59]. In a complete opposite story, ABA is not only found to be a complete inhibitor of GA activity, also a complete inhibitor of AR formation [59]. ABA always remains highest in the non-rooting tissues [60]. But application of IAA attenuates AB and helps in AR formation [61]. Death of epidermal cells overlying the root primordia are important for the emergence of noval adventitious roots which is done by the Programmed Cell Death (PCD). PCD is induced by the ethylene with the help of ROS and also found to be promoted by GA but is blocked by the ABA [60]. It has also been reported that root emergence is dependent on GA 3 activity and concentration while the growth rate of the root depends largely on GA concentration. ABA interferes with the ethylene signaling of root elongation and GA signaling inhibiting root elongation and root emergence [60]. ABA inhibits PCD and that's what may be the reason its concentration got decreased in the fi rst few hours of submergence [60,[62][63][64] and by ten-fold in growth responsive region of deep water rice [60,62,63]. It is also a negative regulator of AR development in tomatoes [65] However, it is also reported that H 2 O 2 mediate the inhibition of root growth in Arabidopsis Columbia by ABA [66].
JA (3-oxo-2-2′-cis-pentenyl-cyclopentane-1-acetic acid), its methyl ester MeJA and isoleucine conjugate (JA-Ile) are considered to be plant stress hormones or endogenous plant growth regulator which comes into play in various stress responses, growth and developmental process [67,68]. They happen to induce stomatal opening, inhibits rubisco biosynthesis, affects nitrogen and phosphorus uptake and transport of glucose and other organic matter [28]. Jasmonate and its methylester, methyljasmonate (MeJA) was found to accumulate rapidly in wounded stems of Soybean and remained elevated up to 24 h and also addition of MeJA up regulated the expression of wound response genes (chalcone synthase, vegetative storage protein, and proline-rich cell wall protein) indicating its major role in wound response [69] and along with ABA, they are suggested to be mediators of wound responses in plants [69]. In contrary fi nding, it has been proposed that auxin pro-  [70][71][72][73]. JA acts as a positive regulator of AR formation in Petunia cuttings [74]. The gene CbNN1 was identifi ed in a study in Catalpa spp, which showed high expression levels in accessions with greater adventitious rooting ability [75]. CbNN1 encodes a WRKY transcription factor and its ortholog in Arabidopsis modulates the crosstalk between the salicylic acid and jasmonic acid (JA) pathways in response to a wide range of biotic and abiotic stimuli [75][76][77].
So, there are many works that supports both the stimulatory and inhibitory effect of ABA, GA, JA and SA in adventitious root formation. But there had been no study in the ROS-PGR interaction. In this study we have tried to explore the effect of redox modulation in SA treated hypocotyl explants on endogenous PGRs GA, ABA and JA and also endogenous ROShormone interaction during the formation of ARF.  After that, the supernatants were thawed adequately at 4° C.

Material and method
The mixture is then extracted with 2.

Spectrofl urometric estimation of total ROS and aspetrophotometric estimation of proxidant (H 2 O 2 )
For the extraction and estimation of total ROS and endogenous H 2 O 2 the process of Kora and Bhattacharjee (2020) [5] was followed.

Statistical analysis
For Average no. of ARF, Results has been calculated as mean of fi ve independent experiments with 15 explants per treatment ± standard error from 5 days old post treatment-raised tissues.

Results
Application of pro-oxidant H2O2 (500 μM) followed bySA (600μM SA) have been shown to signifi cantly augment Adventitious Root Formation (ARF) by 197.2% as compared to untreated control, whereas 10 ×10 -4 M DPI (an inhibitor of NADPH-oxidase) -600 μM SA and 10×10 -4 M DMTU (a free radical scavenger) -600 μM SA combination reduced the formation of ARF in hypocotyl explants of mung bean by 53.5 % and 52.1% (Figure 1,2A,3A). The individual treatment of H 2 O 2 and SA have also found to increase the no. of ARF in hypocotyl explants of mung bean. DMTU and DPI also when treated individually further decreased the no. of AR [5].  It is found to be opposite in relation to these ( Figure 2D,3D).

Discussion
We have earlier reported that H 2 O 2 has signifi cant role in the adventitious root formation in SA treated hypocotyl explants of mung bean [5]. The 500 μM H 2 O 2 -600 μM SA combination has showed greater accumulation of H 2 O 2 and total ROS in compared to the control one. Also where the DPI and DMTU has been used with or without SA, both H 2 O 2 and total ROS found to be greatly reduced suggesting that mung bean explants require ROS signature with a distinct role of redox regulation through antioxidant buffering [5]. The interaction of ROS -PGRs like GA, ABA and JA during ARF has been less studied. There is little direct evidences of the cross talk of ROS and these PGR in ARF.
However, many work have suggested the involvement of ROS, ABA, JA and GA in the signaling pathway initiated by other plant growth hormones like auxin, ethylene etc. [40,48]. In this work, when redox status of SA treated hypocotyl explants of mung bean is manipulated by using pro-oxidant H 2 O 2, NADPH-oxidase inhibitor, DPI and ROS scavenger, DMTU, no clear trend has been found in the endogenous accumulation of these PGRs in different treatment conditions. However, 500 μM H 2 O 2 -600 μM SA combination which has shown signifi cantly higher number of AR have also showed comparatively higher  [5,35]. It is also evident from the results that DPI which inhibits the NADPH-oxidase that gives rise to H 2 O 2 decreased the formation of necessary ROS and hence decreased the ARF.
In the DPI treatment conditions, all of the three PGRs were found to be in lowest concentration indicating that ROS might be necessary for their up-regulation. The results may suggest that ROS, SA and PGRs like ABA, JA and GA acting downstream of other bonafi de plant growth hormones and abiotic stress creating a large complex, sensitive signalling pathway that possess stimulatory and inhibitory effect on morphogenesis of ARF depending on their concentration and time.

Conclusion
Taken as a whole, we can conclude that there lies a complex sensitive hormonal network system where GA, ABA and JA cross talk with ROS in the morphogenesis of AR.  Results are mean of fi ve and three replicates ± standard error for ARF and Total ROS estimation respctively. ** represents signifi cant changes from control at 0.01 level (t-test) and * represents signifi cant changes from control at 0.05 level (t-test).