| Aluminum (Al) toxicity is a major constraint limiting crop growth and yield on acid soils, which occupy approximately 50% of the world’s potentially arable land. Furthermore, more than 60% of the acid soils are located in developing countries, where food security is critical. Inhibition of root elongation is the primary visible symptom of Al toxicity, although Al targets multiple sites in root cells, including plasma membrane, cell wall and cytoplasm.The rapid response of root growth suggests a participation of signal transduction pathways in mediating plant responses to Al toxicity. Polyamines, mainly including putrescine (Put), spermidine (Spd) and spermine (Spm) are ubiquitous signaling molecules in plants and are involved in various physiological processes, such as control of development, senescence, and abiotic stress responses. The endogenous polyamines profiles have been shown to change in plant cells challenged with Al stress; however, the roles of polyamines in the regulation of Al stress responses are unknown.In the present study, we investigated the metabolic changes of polyamines in root tips of wheat, the role of free Put accumulation in Al tolerance strategies, the involvement of H2O2 generated by free Spd oxidation in Al-induced oxidative stress and its regulation by Put, interaction between Put and ethylene in mediating root growth under Al stress, and the possible role of bound Put in Al response by using two wheat genotypes differing in Al resistance (Al-tolerant Xi Aimai-1; Al-sensitive Yangmai-5). Main results are as follows:(1) Using two wheat genotypes with contrasting Al resistance, we studied the effects of Al on polyamine metabolism, reactive oxygen species (ROS) production, oxidative damage and root growth. The results showed that Al caused more Al accumulation. ROS production, oxidative damage and root inhibition in Yangmai-5 than Xi Aimai-1. These were accompanied by different change profiles of endogenous polyamines in two wheat genotypes. Exposure to 30 μM Al elevated total Put content in two wheat genotypes, with the elevation more rapid and conspicuous in tolerant genotype. In contrast, total Spd content exhibited a larger decrease in the sensitive genotype Yangmai-5. Further analyzing the different forms of poly amines indicated that free Put, bound Put levels increased more, while free Spd declined slighter in Xi Aimai-1 than Yangmai-5.Exposure of Al also caused a small increase of conjugated Put, but there is no difference between genotypes. No significant changes were observed in conjugated and bound Spd levels. The accumulation of Put was resulted from increase in arginine decarboxylase (ADC) activity, and inhibitor of ADC significantly aggravated Al-induced oxidative damage. These results suggest that elevation of free Put and its conversion to bound Put are important factors conferring Al tolerance in plants, whereas Spd decreasing may be associated with Al sensitivity.(2) The association of free Put accumulation with Al resistance of wheat was investigated using two wheat genotypes with contrasting Al resistance. Increased Put accumulation by exogenous Put application significantly reduced Al-induced root inhibition and Al accumulation in root tips of wheat seedlings, with the alleviation more pronounced in Yangmai-5; whereas reduced Put synthesis by ADC inhibitor (D-Arg) significantly aggravated Al-induced root inhibition and Al accumulation in Xi Aimai-1, fortunately this could be reversed by concomitant treatment with Put. Modulation of Put level, however, had little effect on malate secretion and rhizosphere pH changes under Al stress. The decrease in Al accumulation in roots after Put application was mainly due to decrease in the amount of Al stored in the cell wall. Al exposure induced more pronounced increase in pectin and hemicellulose, as well as pectin demethylation in the root tips of Yangmai-5 than Xi Aimai-1. Inhibition of Put synthesis by D-Arg exacerbated this effect, especially in Xi Aimai-1; whereas exogenous of Put significantly decreased cell wall polysaccharides and increased the degree of pectin methylation, thus reduced bindings sites for Al. These results suggest that ADC-dependent Put accumulation plays important roles in providing protection against Al toxicity in wheat plants through decreasing cell wall polysaccharides and increasing the degree of pectin methylation, thus decreasing Al retention in the cell walls.(3) The effects of Put and specific enzymes inhibitors (diamine oxidase (DAO) inhibitors 2-bromoethylamine (2-Br) and aminoguanidine (AG); polyamine oxidase (PAO) inhibitors guazatine (GZT) and 1,12-diaminododecane (DADD)) on Al-induced oxidative stress were studied. Exposure to Al caused more serious oxidative damage in Yangmai-5 than Xi Aimai-1. Application of Put and inhibitors of PAO (GZT, DADD) significantly alleviated Al-induced oxidative stress, but not of inhibitors of DAO. Meanwhile, a pronounced increase of cell wall-bound PAO activity and H2O2 production was observed in the root tips of Yangmai-5 than Xi Aimai-1. Treatment of seedlings with Put inhibited the activity of cell wall-bound PAO and partially reduced H2O2 accumulation, same as PAO inhibitors (GZT, DADD) did. Plasma membrane NADPH oxidase was significantly inhibited by exogenous Put, while was not affected by PAO inhibitors. Therefore, our results indicate that Put could alleviate Al-induced oxidative stress though inhibiting cell wall-bound PAO and plasma membrane NADPH oxidase, thus blocked Al-induced H2O2 overproduction.(4) The interaction between Put and ethylene in modulating Al-induced root inhibition was investigated using Put and different ethylene donors and inhibitors. Aluminum caused more root inhibition in Yangmai-5 and increased ethylene production at the root apices compared to Xi Aimai-1, whereas the effects were significantly reversed by inhibitors of ethylene biosynthesis. The simultaneous exposure of wheat seedlings to Al and ethylene donor, ethephon, or ethylene biosynthesis precursor,1-aminocyclopropane-1-carboxylic acid (ACC), promoted ethylene releasing and aggravated root inhibition, which was more pronounced in Xi Aimai-1. In contrast, Put treatment reduced ethylene production and mitigated Al-induced root inhibition in both genotypes, and the effects were more pronounced in Yangmai-5. Furthermore, our results indicated that Al-induced ethylene production was mediated by ACS synthase (ACS) and ACC oxidase (ACO), and that Put decreased ethylene production by inhibiting ACS. Altogether, these findings indicated that ethylene is involved in Al-induced root inhibition and this process could be alleviated by Put through inhibiting ACC activity.(5) The possible role of Al-induced elevation of bound Put in regulating Al tolerance was investigated by adding the specific inhibitor of bound Put synthesis (phenanthrolin, o-phen). A significant increase of bound Put was observed in the root tips of Al-tolerant genotype Xi Aimai-1, whereas the Al-sensitive genotype Yangmai-5 showed no bound Put accumulation under Al stress. Treatment of seedlings with o-phen abolished Al-induced bound Put accumulation in the root tips of wheat seedlings. Exposure to Al caused more serious root inhibition and callose accumulation, as well as ROS accumulation and oxidative damage in Yangmai-5 than Xi Aimai-1. Eliminating bound Put accumulation in Xi Aimai-1 with o-phen exacerbated the Al-induced ROS accumulation and oxidative stress. Under Al stress, more significant increase in SOD, CAT, APX activities were observed in Xi Aimai-1 than Yangmai-5; however, this increase in Xi Aimai-1 was suppressed by o-phen treatment. These results indicated that Al-induced bound Put plays an important role in Al resistance of wheat through maintaining enhanced activities of antioxidant enzymes and thus eliminating ROS accumulation, alleviating Al-induced oxidative damage. |
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