Waterlogging stress in plants evokes physiological, morphological and molecular level changes. Waterlogging conditions first influence the root; the adventitious root development and aerenchyma emergence during waterlogging stress is an essential adaptive trait. In this study, we have tried to understand the mechanism behind the waterlogging tolerance in M.arvensis by comparing the control root (primary tap root) and waterlogging-induced adventitious root (8 days of waterlogging treatment). The adventitious root contains a more significant number of aerenchyma that not only facilitates gas transport from shoots to roots but also decreases the number of cells, which consumes more oxygen (Mano Y & Omori F. 2013).
Waterlogging results in extensive transcriptome profiling, and we investigated the differential gene expression pattern. After removing adaptor or vector-containing sequences, we obtained 25.325 million reads in the adventitious root and 20.95 million reads in the control root. After de novo assembly, we obtained 219957 and 162193 high-quality transcripts unique to the adventitious root and transcripts in the control root.
4.1 Transcriptome-based differential expression profiling provides a genetic basis for the adaptive response of plants after waterlogging:
In the context of global climate change, waterlogging emerges as a serious concern affecting the worldwide productivity of crops. Different cellular and metabolic changes occur inside the plant system to overcome this condition, which makes them necessary to understand to make them tolerant under these circumstances. Plants undergo morphological and anatomical changes during this stress period along with cellular level changes. For example, in cucumber, waterlogging induced the accumulation of ethylene, resulting in the formation of ARs (Qi et al. 2020). Aerenchyma emergence in ARs is an adaptable response resulting from programmed cell death regulated by hormones, mainly ethylene, triggered in many steps. Comparative RNA-seq-based transcriptome analysis between waterlogging tolerant and sensitive varieties of cucumber (Xu et al. 2017) and Chrysanthemum morifolium (Zhao et al. 2018) broadens our knowledge regarding the molecular mechanism of waterlogging response in plants. High-throughput RNA sequencing was also adopted for elucidating the functional pathway and regulatory gene network of the waterlogged root system of maize (Arora et al. 2017) and Jatropha (Juntawong et al. 2014). Comparative functional enrichment and gene ontology study of the root transcriptome of these species demonstrated the physiological response promoted by waterlogging treatment. In this study, we have made a comparative de novo transcriptome analysis of the control root vs 8th day waterlogging induced adventitious root of M. arvensis. This transcriptome analysis has helped to identify differentially regulated transcripts involved in waterlogging-induced adventitious root formation, which has not been extensively studied. DEG data shows among 208081 unigenes, 47534 unigenes were upregulated, 36037 unigenes were downregulated and 124510 unigenes were neutrally regulated in adventitious root. This indicates the differential gene expression responsive to waterlogging. Gene ontology enrichment analysis grouped transcripts into three domains biological process, cellular components and molecular functions (Fig. 4 and S5). Transcripts in response to hormonal regulation, carbohydrate metabolism, antioxidant activities, and regulation of transcription in adventitious roots were searchedd. Based on the previous literature, transcripts related to these metabolisms may be involved in providing waterlogging tolerance to the plant.
4.2 Regulation of hormone in waterlogged M. arvensis adventitious root emergence:
Ethylene is the primary factor that induces adventitious root formation during waterlogging stress (Sasidharan et al. 2015), and it was observed that ethylene biosynthetic inhibitor (CoCl2) suppresses adventitious root formation in M. arvensis by inhibiting ACC oxidase, an important enzyme of the biosynthetic pathway of ethylene (Phukan et al.2014).In the current study, twelve transcripts of S-adenosylmethionine, an intermediate of ethylene signalling, were explicitly accumulated in the adventitious root. Ethylene also regulates the hormone signalling of abscisic acid, gibberellic acid, and eventually auxin that, leads to adventitious root formation in tomatoes, Solanum dulcamarum, and Oryza sativa (Steffens et al. 2006; Dawood et al. 2016). Auxin accumulation in the plinth of the stem promotes adventitious root formation. It was observed that in the tomato plant, adventitious root formation depends upon auxin concentration, whereas in the tomato plant, in which auxin sensing is compromised, the emergence of the adventitious root is prohibited (Vidoz et al. 2010). There were 4 transcripts related to auxin present in the M.arvensis adventitious root. Our transcriptome data showed the accumulation of 7 ABA transcripts during the waterlogging; this can be correlated with ABA form increased extensively as the period of flooding progressed (Zhang et al. 1987). During waterlogging onset, plant roots promptly mediate xylem-borne stimulus to leaves via hormone, most prominently abscisic acid (ABA), which slows transcription via the complete closure of stomata (Jackson, 2005).In the pea (pisum sativum), after 4 days of flooding, it was observed that the abscisic acid (ABA) level was upregulated in the roots, resulting in complete stomatal closure.
In our study, transcripts belonging to the hormonal group of auxin, ethylene and abscisic acid biosynthesis, transport and metabolism were found to be upregulated in the adventitious root, indicating that these groups of hormone acts as a central regulator of waterlogging response and adventitious root formation. We have also found that 12 and 3 transcripts related to hormone biosynthesis and cascade were upregulated in adventitious roots related to cytokinin and strigolactone biosynthetic processes. Ethylene and abscisic acid-related transcripts were majorly found to be upregulated in the adventitious root, as it has already been reported their signalling cascade increases and implicates the emergence of the adventitious root (Vidoz et al., 2010) (Fig. 6d and 7d).
4.3 Effect of waterlogging on regulatory transcription factor:
Transcription factors (TFs) modification activity dynamically changes the transcriptome, which causes metabolic and phenotypic changes (Mitsuda et al. 2009). Transcriptional regulation in waterlogging conditions (hypoxic) has been extensively elucidated in the dicot model plant Arabidopsis thaliana. Transcription factor family of MYB, NAC, and AP2/ERF are shown to affect hypoxia responses in Arabidopsis (Hsu et al. 2011). As discussed earlier, ethylene has a significant role in adventitious root formation during waterlogging, and an ERF transcription factor regulates ethylene signal transduction (Licausi et al. 2013). The main transcription factor that regulates the hypoxia-related gene expression and responses to waterlogging conditions is Group VII ERFs (Mizoi et al.2012). In Jatropha, JcERFVII-2 and JcERFVII-3, were two ethylene response factors (ERFs) that were found to induce in response to waterlogging (Juntawong et al. 2014). In addition to AP2/ERF, a MYB transcription factor of Triticum aestivum i.e., TaMyb1 is vigorously induced in the roots under the hypoxic condition (Lee et al. 2007). In our data, most transcripts related to the transcription factor were involved and expressed in the adventitious root. The top five transcription factor families showing upregulated expression in adventitious root after waterlogging include C3H zinc finger, Orphan, mTERE, MYB related, FAR1, AP2/ERBP family of transcription factor. The determination of 54 families of transcription factors are either uniquely expressed or upregulated in adventitious root indicates a tight regulation and cross-talk of different signalling pathways during waterlogging.
4.4 Effect of waterlogging on antioxidative defence system:
Adventitious root growth is enhanced by both hormonal signals and reactive oxygen species. Ethylene by the plasma membrane-located NADPH oxidase enhances superoxide anion generation in the flooded rice. Accumulation of ROS augments root development by suppressing antioxidant enzyme catalase (with aminotriazole), whereas scavenging of hydrogen peroxide (with potassium iodide) suppresses the growth (Steffens et al., 2012), which indicates the hydrogen peroxide significance in crown root growth (Steffens et al., 2006). Some of the antioxidant enzymes involved in detoxification are superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidases (APX). Similarly, transcripts belonging to the catalase, peroxidase and superoxide dismutase were found to be upregulated in adventitious root in our transcriptome data, also after performing the biochemical activity followed by different days of waterlogging treatment antioxidant enzymes like catalase, SOD and NO scavenging activity was markedly induced in adventitious root in comparison with control root indicating that either they are involved in adventitious root development or plant is coping up under stressful environment after waterlogging treatment.
4.5 Waterlogging also affects energy-consuming metabolic processes:
The availability of carbohydrates, their metabolism and transport are necessary to regulate waterlogging and multiple abiotic stress tolerance (Rosa et al., 2009). In deep waterlogging conditions and if waterlogging persists long, the carbohydrate availability from the photosynthetic process or stored food reserve becomes crucial. Therefore, during the above-discussed waterlogging conditions, plants must alter the survival mechanism involving carbohydrate consumption (Bailey-Serres et al. 2008). It was reported that gene expression of α-amylase (OsAmy3D) in submerged rice leaves is modulated by SUB1C, an ethylene-responsive factor (ERF) (Fukao et al., 2006). Gibberellic acid positively regulates this gene and negatively by a related ERF SUB1A1. These findings suggest that carbohydrate levels in submerged plants are under hormone control. Glycolytic genes in S. dulcamara were enhanced after 6 h of submergence, demonstrating metabolic acclimatization to waterlogging stress (Dawood et al. 2013). Metabolic mechanisms related to carbohydrate mobilization in Cucumis sativus L. are related to its capacity to face oxygen deprivation and generate adventitious roots (Xu et al., 2018). Recent experiments suggest that flood-resistant and sensitive plants have different abilities to maintain their carbohydrate content during waterlogging (Vu &Yelenosky 1991; Ferner et al. 2012). Plants which are more waterlogging sensitive their soluble sugar level deplete faster as compared to tolerant varieties as they were able to maintain a high level of total carbohydrates during the stress (Ferner et al. 2012); this correlates with our data where we found carbohydrate level was more in waterlogging induced adventitious root as compared to the control root (Fig. 3A). Reserve starch breakdown can partially fulfil the carbohydrate demand in the waterlogging tolerant species (Kreuzwieser et al. 2014). During waterlogging stress, carbohydrate metabolism is activated in the root system to produce more glucose and maintain energy levels (Zhang et al. 2017). Waterlogging-responsive AP2/ERF from M. arvensis MaRAP2-4 is shown to regulate AtSWEET10 in Arabidopsis thaliana, leading to stress response (Phukan et al. 2018). In our transcriptome data, enzymes related to carbohydrate metabolism like pyruvate kinase, fructose 6- phosphotransferase, glyceraldehyde-3-phosphate dehydrogenase etc., were found to be upregulated in waterlogging-induced adventitious root along with that the total carbohydrate content of adventitious root is found to be higher than control root suggesting their involvement in energy generation process required during the adventitious root generation under waterlogging.