Cytokinin and gibberellic acid-mediated waterlogging tolerance of mungbean (Vigna radiata L. Wilczek)

Introduction

Mungbean (Vigna radiata L. Wilczek) is considered a major source of pulse protein because of its high protein content in the seeds (20.97–31.32%) (USAID Agricultural Extension Support Activity, 2017; (Yi-Shen, Shuai & FitzGerald, 2018; Rahman et al., 2019). More fundamentally, the mungbean is gaining its popularity in the extensive rice-based cropping system, owing to its low fertilizer and pesticide requirements, which provides farmers with good economic benefits and nutritional security (DAE, 2018; Rahman et al., 2019; Hou et al., 2019; Bangladesh Water Development Board, 2020).

Waterlogging has become a major environmental problem in global agriculture (Kaur et al., 2020). Under waterlogged conditions, the mass of oxygen in the soil attenuates partially (hypoxic) or fully (anoxic) due to high microbial activity, and the resulting CO₂ accumulation in the root zone restricts root metabolism, aerobic respiration, ATP synthesis, and nutrient acquisition, resulting in a significant reduction in the growth, development, and biomass of roots and shoots (Najeeb et al., 2015; Gill et al., 2018; Dossa et al., 2019). Moreover, waterlogging impairs root permeability and causes root injury, leading to a decrement of hydraulic conductivity and consequent stomatal closure, resulting in a significant reduction in net photosynthetic and transpiration rates (Else et al., 2001; Anee et al., 2019). These also lead to the excess generation of reactive oxygen species (ROS) such as superoxide (O₂^•−), singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂) and hydroxyl radical (^•OH), which are harmful to biological macromolecules (Yeung et al., 2018; Zhou et al., 2020).

Plants can acclimatize to the adverse impact of waterlogging-mediated soil oxygen dearth through adapting various morphological, physiological, and biochemical mechanisms. The development of adventitious roots and faster stem elongation are some examples of morphological adaptations (Yamauchi et al., 2018), whereas reduced stomatal conductance and subsequent decrease in net photosynthetic rate are short-term physiological adaptations (Jacobsen et al., 2007; Bhusal et al., 2020). Additionally, to shield themselves from stress, waterlogged plants accumulate several osmoprotectants such as proline, soluble sugars, and sucrose (Tewari & Mishra, 2018). However, one of the most important adaptive mechanisms is a well-balanced antioxidant defense system that involves both enzymatic (e.g., superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; glutathione S-transferase, GST; glutathione peroxidase, GPX) and non-enzymatic (e.g., total phenolics, total flavonoids, carotenoids) antioxidants that scavenge overaccumulation of ROS (Garcia et al., 2020; Hasanuzzaman et al., 2020).

Since mungbean is a waterlogging sensitive crop, there must devise a strategy for effectively alleviating the destructive effect of waterlogging stress to improve mungbean production in flood-prone areas having intensive and erratic rains. In this context, the search for effective approaches propelled us to use phytohormones such as cytokinin (CK) and gibberellic acid (GA₃) for overcoming the problems associated with waterlogging.

Although there are several studies on the role of phytohormones on abiotic stress tolerance in crop plants, there are hardly any studies focusing on the coordinated actions of CK and GA₃ in modulating plant growth and physiology. In the present study, we examined whether CK and GA₃ protect mungbean from waterlogging damage. We also examined the regulatory roles of CK and GA₃ in improving waterlogging tolerance of mungbean by investigating the morpho-physiological and biochemical mechanisms through assessments of morpho-physiology, photosynthetic parameters, ROS metabolism, osmoregulation, and yield.

Materials and Methods

Results

Exogenous CK and GA₃ enhanced gas exchange features of mungbean plants during waterlogging stress and recovery period

Plants subjected to waterlogging stress and subsequent recovery period showed a decrease in Pn (by 44% and 42%), gs (90% and 53%) and E (74% and 36%, respectively) when compared with control plants (Figs. 1A–1C). Nonetheless, the levels of WUEint and WUEins in WL plants increased by 515% and 118%, respectively relative to control plants, whereas the level of WUEint and WUEins in control and WL plants was comparable after the recovery period (Figs. 1D, 1E). On the other hand, CK+WL and GA₃+WL plants exhibited decreased levels of gs (by 40% and 27%) and E (33% and 23%), and increased levels of Pn (by 37% and 27%), WUEint (119% and 64%) and WUEins (100% and 61%, respectively) when compared with that of WL plants (Figs. 1A–1E). Similarly, following the recovery period, decreased levels of gs (by 36% and 28%) and E (34% and 21%), and increased levels of Pn (37% and 27%), WUEint (115% and 73%), and WUEins (107% and 59%) were recorded in CK+WL and GA₃+WL plants, respectively, as compared with that of WL plants (Figs. 1A–1E).

CK and GA₃ prevented damage to photosynthetic pigments during waterlogging stress and recovery period

Compared to control plants, a sharp decline in the contents of Chl a (by 50.10% and 43%), Chl b (65.44% and 63%), and Chl (a+b) (54% and 49%) were observed in the leaves of WL plants following stress and recovery periods, respectively (Figs. 2A–2C). In contrast, CK and GA₃ supplementation protected the photosynthetic pigments from waterlogged-induced deleterious effects by enhancing the contents of Chl a (by 74% and 61%), Chl b (142% and 124%), and Chl (a+b) (88% and 74%) in the leaves of CK+WL and GA₃+WL plants, respectively (Figs. 2A–2C). Furthermore, following the recovery period, the leaves of CK+WL and GA₃+WL plants also displayed a significant rise in the content of Chl a (by 60% and 50%), Chl b (134% and 120%), and Chl (a+b) (75% and 64%, respectively) in comparison with WL plants (Figs. 2A–2C).

Exogenous CK and GA₃ suppressed oxidative stress markers in mungbean plants during waterlogging stress and recovery period

Imposition of waterlogging stress and following recovery period substantially increased the contents of H₂O₂ (by 113% and 62%) and MDA (179% and 100%, respectively) in the leaves of WL plants, relative to that of control plants (Figs. 3A, 3B). CK and GA₃ treatment resulted in reductions of H₂O₂ (by 57% and 45%) and MDA (29% and 19%) contents in the leaves of CK+WL and GA₃+WL plants, respectively, in comparison with WL plants (Figs. 3A, 3B). Similarly, compared with the WL plants, substantial decreases in the contents of H₂O₂ (by 37% and 26%) and MDA (34% and 21%) were noticed in the leaves of CK+WL and GA₃+WL plants, respectively, in the recovery period (Figs. 3A, 3B).

Discussion

Waterlogging leads to detrimental consequences on a number of crop plants, including mungbean (Amin et al., 2015; Donat et al., 2016). The current study investigated the effective mitigation of waterlogged-induced damage to mungbean plants by exploring the potential role of CK and GA₃.

In the present study, waterlogging-induced deleterious effects were evident as a reduction of shoot height, shoot and root DW, and stem girth; however, the foliar application of CK and GA₃ partially alleviated those waterlogged-mediated detrimental effects. It is well reported that both CK and GA₃ played a pivotal role in cell elongation and other growth and development processes (Ahanger et al., 2018; Wang et al., 2019; Rady et al., 2021), which resulted in enhanced growth and biomass of mungbean under waterlogging in the present study.

The waterlogged-induced growth inhibition and biomass reduction in mungbean plants might be a consequence of impeded photosynthesis. It is envisaged that at the beginning of waterlogging stress, plant roots rapidly transmit a xylem-borne signal to the leaves in the form of hormones, most notably abscisic acid (ABA) to slow down the process of transpiration through stomatal closure, and thus attenuate carbon dioxide availability in leaves (Jackson et al., 2003; Najeeb et al., 2015). This was due to the decline in root hydraulic conductivity and inadequate ATP production during waterlogging as reported by other researchers (Steffens et al., 2005; Kaur et al., 2019). Henceforth, the supply of photoassimilates from leaves to different plant parts is also reduced, which ultimately leads to poor plant growth and biomass production (Kogawara et al., 2006). Apart from these, waterlogged-induced reduction of photosynthetic pigments, including Chl a and b, is also responsible for reduced net photosynthetic rate (Ren et al., 2016; Zhang et al., 2020), as also observed in the present study (Figs. 1A and 2A–2C). On the other hand, mungbean plants treated with CK and GA₃ significantly improved the net photosynthetic, as well as the contents of Chl a and Chl b under waterlogged conditions, implying that CK and GA₃ played a pivotal role in improving the photosynthetic process during waterlogging stress. Improvements in photosynthesis, as well as photosynthetic pigments, were also observed after the recovery period in response to CK and GA₃ supplementation. This was due to the fact that phytohormones could regulate photoprotection of the photosynthetic apparatus under stress conditions and they controlled the production and scavenging of photosynthesis-derived ROS, the duration and extent of photo-oxidative stress, and redox signaling (Müller & Munné-Bosch, 2021).

We also observed that in comparison with WL plants, CK or GA₃-treated plants demonstrated remarkably improved WUE, indicating that CK and GA₃ might enable mungbean plants to produce more biomass under the conditions of limited water uptake. Surprisingly, after the recovery period, we have seen similar positive effects of CK and GA₃ in boosting WUE. It is well-known that improving WUE in abiotic-stressed plants without deteriorating growth and yield is scrutinized as a paramount goal of current plant breeding programs (Yang et al., 2016). The increase in WUE was mainly due to the enhancement of photosynthetic parameters. Our findings support this phenomenon and corroborate with the previous findings where the external application of CK to cold-stressed coffee (Coffea arabica) and GA₃ to drought-stressed Vicia faba seedlings improved WUE (Acidri et al., 2020; Rady et al., 2021).

Water stagnation because of waterlogging conditions causes poor gas exchange between soils and plant roots, resulting in hypoxia or anoxia in plant tissues (Matin & Jalali, 2017). The scarcity of oxygen induces leakage of electrons from mitochondria, which together with impaired photosynthesis, triggers a burst of excessive ROS production, causing oxidative damage to waterlogged-stressed seedlings (Xu et al., 2013), and was manifested in our study with increasing H₂O₂ and MDA in WL plants (Figs. 3A, 3B). External application of either CK or GA₃ to waterlogged-stressed mungbean plants resulted in a dramatic reduction in ROS accumulation, implying that CK and GA₃ play an important role in reducing oxidative burden induced by ROS and providing protection to plasma membrane integrity. It is well established that phytohormones controlled the production and scavenging of photosynthesis-derived ROS and also enhanced antioxidant defense (Müller & Munné-Bosch, 2021). In line with our study, previous reports also demonstrated that the contents of H₂O₂ and MDA were noticeably diminished in salt-stressed tomato (Solanum lycopersicum) and okra (Abelmoschus esculentus) by CK application, and drought-stressed wheat (Triticum aestivum) and boron-stressed tomato by GA₃ application (Ahanger et al., 2018; Wang et al., 2019; Moumita et al., 2019; Javed et al., 2021).

Our results also demonstrated that, compared with WL, the leaves of CK+WL and GA₃+WL plants accumulated higher levels of non-enzymatic antioxidants, namely total phenolics and total flavonoids (Figs. 3E, 3F). More fundamentally, following the recovery period, there was a similar pattern of improvement in total phenolics and total flavonoids levels in response to CK or GA₃ application. Phenolics and flavonoids are two well-known secondary metabolites that play a pivotal role in scavenging free radicals and preventing lipid peroxidation, thus maintaining membrane fluidity and shielding cell membrane damage from oxidative damage under different abiotic stresses, including waterlogging (Ahmad et al., 2010; Patel et al., 2020; Elkelish et al., 2020). These results implied that either CK or GA₃-induced alleviation of ROS burden was most likely due to the heightened level of non-enzymatic antioxidants. It is worth noting that the application of CK to mungbean plants displayed a greater role in alleviating the contents of H₂O₂ and MDA by enhancing the levels of total phenolics and flavonoids when compared with the plants treated with GA₃. Ahanger et al. (2018) reported that CK application to salt-stressed tomato plants increased the level of total phenolics and flavonoids, while Rady et al. (2021) revealed that GA₃ supplementation to drought-stressed faba bean plants increased phenolics accumulation.

To overcome waterlogged-induced osmotic stress, plants also accumulate several compatible solutes, including proline and total soluble sugars, which play vital roles in maintaining water balance, retrieving photosynthetic functions, stabilizing cellular components, ROS scavenging, improving cellular signaling, and secondary metabolite biosynthesis (Barickman, Simpson & Sams, 2019; Chávez-Arias, Gómez-Caro & Restrepo-Díaz, 2019; Xiao et al., 2020). Our data on compatible solutes revealed that the levels of proline and total soluble sugar were dramatically enhanced in WL plants (Figs. 3C, 3D). Importantly, exogenous application of CK and GA₃ further enhanced the contents of proline and total soluble sugars in the leaves of CK+WL and GA₃+WL plants, and this trend was similar following the recovery period (Figs. 3C, 3D). Our findings coincided with the results of Sarafraz-Ardakani et al. (2014), who also observed that CK supplementation increased the amount of proline and soluble sugars in drought-stressed wheat plants.

Waterlogged-stressed mungbean plants showed a decrease in yield-associated features such as pod length, the total number of pods per plant, thousand seed weight, and seed yield per plant, which might be correlated with poor plant growth because of impaired photosynthesis and enhanced accumulation of ROS (Kumar et al., 2013). In contrast, foliar application of either CK or GA₃ to waterlogged-exposed mungbean plants resulted in a dramatic increase in pod length, the total number of pods per plant, thousand seed weight, and seed yield per plant. These results suggested that CK or GA₃ supplementation improved photosynthetic efficiency of mungbean to sustain higher levels of total soluble sugars, as well as heightened levels of proline and non-enzymatic antioxidants to resist the phytotoxic effects of waterlogging through the reductions of ROS-mediated oxidative stress, resulting in improvement of yield performance. Yield improvement in wheat plants under heat and drought stress was also reported upon supplementation of CK and GA₃, respectively (Gupta, Agarwal & Gupta, 2012; Yang et al., 2016).

Conclusions

Our work revealed that treating plants with either CK or GA₃ positively regulates defense responses in waterlogged-stressed mungbean plants through regulating several physiological and biochemical mechanisms. The CK and GA₃-sprayed plants demonstrated improvement in plant height, stem girth, and plant biomass and a decreased level of ROS accumulation and consequent MDA level, implying that CK and GA₃ played a decisive role in controlling ROS-induced cellular damage. These observations were closely interlinked to heightened levels of non-enzymatic antioxidants, including total phenolics and total flavonoids. Elevated levels of proline and total soluble sugars in CK and GA₃-sprayed plants also contributed to the maintenance of better water status and osmotic adjustment under waterlogging. Such positive regulatory roles of CK and GA₃ also persisted after the 15-day recovery period. All the studied physiological and biochemical mechanisms in CK and GA₃-sprayed plants contribute to improving the yield-associated features of mungbean plants.

These findings imply that CK and GA₃ could be used as a potential chemicals for reducing the negative impacts of waterlogging on crops and on agricultural production to ensuring the sustainability of agriculture in flood-prone areas.

Supplemental Information