As aerobic organisms, plants need oxygen (O2) to support respiration, metabolism and growth. Plants frequently suffer from hypoxic stress due to the low O2 concentration that is caused by long-term flooding, waterlogging, soil compaction or soil cover management (Bailey-Serres and Voesenek, 2008, Xu et al. 2017). Hypoxia triggers plant physiological changes and gene expression (Gibbs et al. 2011). In our study, flooding was shown to inhibit the leaf length and leaf area in Phyllostachys praecox, as expected. Exogenous Spd alleviated bamboo growth inhibition with flooding (Fig. 1). This was consistent with the result that Spd alleviated the inhibition of soybean seedling growth under excess soil moisture (Sidhu et al. 2020).
Generally, membrane lipid peroxidation increases significantly (P<0.05) with time under flooding (Yiu et al. 2009). In this study, the MDA content increased with time, suggesting that the flooding treatment enhanced lipid peroxidation. The application of Spd reduced the MDA content, indicating that Spd can reduce the oxidative stress caused by flooding (Fig. 2, Table 2). This is in accordance with that reported by Hussain et al. (2019), where Spd reduced MDA content and ROS concentrations in Brassica juncea leaves.
SAMDC is the rate-limiting enzyme in the synthesis of Spd and Spm in plants (Mehta et al. 2002). Studies have reported that after 6 h of drought stress, the PA content in CaSAMDC-overexpressing transgenic Arabidopsis increased, and the accumulation of ROS in cells decreased significantly (Wi et al. 2014). In the present study, flooding induced an increase in SAMDC activity after 2 d. In the middle and late stages of the experiment, SAMDC activity decreased significantly (P<0.05) under flooding (Fig. 3a, Table 2). The transient increase in SAMDC activity may lead to an increase in PA content to protect bamboo from flooding stress. However, as the degree of stress increases, a large amount of ethylene is synthesized in plants. Therefore, it is possible that the concentrations of Spd and Spm that share a common precursor (SAM) with ethylene decreased, and the activity of SAMDC therefore decreased. We found that after exogenous Spd application, the SAMDC activity increased significantly (P<0.05) and the ethylene biosynthesis rate-limiting enzyme activities and gene expression decreased. This may be because exogenous Spd led to an increase in the content of endogenous Spd and Spm in bamboo. The increase in SAMDC activity may accelerate the conversion of free Put to Spd and Spm, and then the conversion of free Spd to Spm, while reducing the ethylene content. This hypothesis is consistent with previous studies, where Hu et al. (2012) found that exogenous Spd significantly improved Spd and Spm content, and enhanced SAMDC activity under salt stress.
Nitric oxide plays a key role as an intra- and intercellular messenger, inducing various processes in plants, including the expression of related genes and programmed cell death, stomatal closure, seed germination, cadmium toxicity and root development (Wendehenne et al. 2001, Angélique Besson-Bard and Wendehenne, 2009). The source of NO in plants is very rich, and it is mainly produced through the activities of NO synthase (NOS) and nitrate reductase (NR). NR is a cytosolic enzyme that catalyzes NADH-dependent nitrate reduction into nitrite. Nitrate reduction may contribute to cellular acclimation to low oxygen deprivation by regenerating NAD+ from NADH. Accordingly, species tolerant to oxygen deprivation exhibit higher NR activity than sensitive ones (Bailey-Serres and Voesenek, 2008). In the present study, we found that flooding significantly (P<0.05) improved the NR activity of leaves (Fig. 3b, Table 2). Both in tobacco and in tomato, it has been shown that the absence or the decrease in NR activity in transgenic plants or the addition of tungstate (a potent inhibitor of NR activity) enhances the symptoms of hypoxia. These symptoms are accompanied by a reduction in plant growth (Stoimenova et al. 2003, Horchani et al. 2010). We also found that exogenous Spd decreased NR activity under flooding conditions. Furthermore, with the increase of Spd concentration, NR activity decreased significantly (P<0.05) (Fig. 3b, Table 2). It might be possible that PAs promote the interaction between NO and 14-3-3 proteins to inhibit NR (Rosales et al. 2012).
Polyamines are often regarded as second messengers of plant growth regulators or plant hormones (Tassoni et al. 2000). S-adenosylmethionine (SAM) is a common precursor in the biosynthesis pathway of polyamines and ethylene. Therefore, there may be different interactions between polyamines and ethylene in cells (Ning et al. 1992). One possible interaction is the mutually antagonistic relationship, and the other is that there is no antagonistic relationship between the two. ACS and ACO activities are generally the rate-limiting step in the ethylene biosynthetic pathway (Xu and Zhang, 2015). In the present study, we found that flooding significantly (P<0.05) improved enzyme activities and expression of ACS and ACO, which were significantly (P<0.05) down-regulated by Spd (Fig. 6, Table 3). Moreover, ACS is more sensitive to flooding stress than ACO (Table 3). It indicates that ethylene and polyamines may have an antagonistic effect under flooding. These results are similar to those of previous studies (Rieu et al., Yu et al. 2014). Polyamines are involved in the regulation of the expression of ACS (Pathak et al. 2014). The production of ethylene can be affected by the regulation of ACC synthase and ACC oxidase, at the same time, ethylene can also affect the amount of polyamines in tissues by inhibiting the activity of polyamine synthases such as ADC (Ning et al. 1992).
With ABA, studies have shown that ABA has a certain inhibitory effect on the content of polyamines in plant tissues (Mahajan and Sharma, 2009, Guo et al. 2018). Under water-deficient conditions, the endogenous ABA content from roots to leaves increased and induced stomatal closure (Matías et al. 2015). On the other hand, Luo et al. (2019) suggested that the promoting effect of external Spd on grain filling of wheat was significantly related to the increasing concentration of ABA in grains. The ABA response to flooding may differ and depend on the plant species and duration of flooding. Under normal circumstances, ABA mainly exists in the chloroplast membrane of mesophyll cells by binding to proteins. When the plant is under stress, the bound ABA is quickly released and changes into a free state, which increases the content of ABA in the plant (Sembdner et al. 1980). The results of the present study showed that ABA content increased in the early stages of flooding. This showed that ABA responds quickly in the initial stage of flooding stress, and mainly protects the cell structure and function of bamboo. Exogenous Spd increased ABA content under flooding stress in the initial stages. With the increase of exogenous Spd concentration, the ABA content also increased (Fig. 5a, Table 2). This is consistent with previous studies. Tajti et al. (2019) suggested that PAs induced ABA accumulation in Spd-treated plants. In our study, after 8 d flooding, ABA content of flooded plants significantly (P<0.05) decreased compared with controls (Fig. 5a, Table 2). This is probably due to the increase in membrane permeability and cell function damage by the prolonged flooding time. The rate of ABA catabolism was higher than its rate of synthesis, which led to a decrease in the ABA content. Wang et al. (2010) also found that after 15 d of flooding, the ABA content decreased in squash trees.
It is well known that IAA can promote plant growth. At the beginning of flooding, the content of IAA increased in bamboo tissues. The increase of auxin content at the early stage of flooding is beneficial to the initiation of stem elongation, leaf growth and oxygen transport (Spanu et al. 1994, Eysholdt-Derzso and Sauter, 2017). Similarly, we found that IAA content increased during 2-4 d of flooding. However, we also noted a significant (P<0.05) decrease in IAA content on the sixth and eighth day after flooding (Fig. 5b, Table 2). This is probably because in the late stages of flooding, photosynthesis of P. praecox was severely blocked, membrane lipid peroxidation was severe, and cellular structures were destroyed, thus, the bamboo could not provide energy and substances to meet the needs of IAA synthesis. In our study, exogenous Spd significantly (P<0.05) increased IAA content of bamboo under flooding (Fig. 5b, Table 2). This may be a protection mechanism for plants to adapt to the flooded environment. Li et al. (2018) suggested that drought stress significantly increased the ABA, methyl jasmonate (MeJA) and salicylic acid (SA) concentrations, and notably decreased the IAA, gibberellins (GA3) and zeatin-riboside (ZR) concentrations in maize seedlings.
Many auxin-related genes participate in plant development by regulating the auxin balance in processes such as cell division and elongation, morphogenesis of roots and stems, apical dominance, and plant leaf bud and fruit development (Chandler and William, 2016). Some genes, such as auxin/indole-3-Acetic Acid (Aux/IAA) are responsive to auxin stimulation in the early stage of auxin signal transduction (Chapman and Estelle, 2009). Auxin signaling involves the regulation of gene expression by Auxin Response Factors (ARFs) and their inhibition by Aux/IAA proteins (Ori, 2019). ARFs can initiate or inhibit the expression of primary early auxin response genes by specifically binding to the auxin response elements (AuxREs) in the promoter part (Shin et al. 2007), and participate in different growth processes of plants. In Arabidopsis, the ARF protein family is divided into two categories: transcription activator and transcription repressor (Tiwari and Guilfoyle, 2003). So far, only ARF2, ARF3, ARF4 and ARF9 proteins have been proved to have transcriptional inhibition through plant protoplast transformation experiments (Ulmasov, 1997).
There is evidence that the expression of ARF is affected by environmental or hormonal signals. For example, as the degree of leaf senescence deepens, the expression of ARF2 increased, while that of ARF1 decreased in Arabidopsis leaves (Ellis et al. 2005). Similarly, we found that flooding significantly (P<0.05) decreased ARF1 expression of leaves and Spd application up-regulated ARF1 gene expression under flooding (Fig. 7e, Table 3). It was suggested that ARF1 was likely to be a transcription activator. It has been reported that AUX1 is an auxin uptake carrier (Marchant, 2014). AUX1 could directly be involved in induction of ROS signaling via the H2O2-mediated pathway, which prevents further increase in oxidative damage. Alternatively, AUX1 could indirectly influence cell elongation and cell division by regulating auxin levels and the auxin signaling network, in turn controlling the root growth under stress (Krishnamurthy and Rathinasabapathi, 2013). The present study showed that the expression of AUX1, AUX2, AUX3 and AUX4 genes of bamboo under flooding was significantly (P<0.05) reduced, and AUX1, AUX2 and AUX4 were more sensitive to flooding (Table 3), while exogenous Spd up-regulated the expression of these genes (Fig. 7a-d, Table 3). This may be due to the synergistic effect of spermidine and IAA to alleviate the damage caused by flooding stress.