The Mechanism Of Phytic Acid Dgradation Mediated By Polyamines And Ca²⁺ In Mung Bean Sprouts

Phytic acid is usually deposited as phytateemineral and phytate-mineral-protein complexes which are not absorbed by monogastric animals,resulting in poor bioavailability of micronutrients.Seed germination used to activate endogenous phytase for degrading phytic acid content can not only improve the bioavailability of mineral nutrients,but also enhance the formation of lower inositol phosphates which have anti-oxidation,anti-cancer;blood pressure-lowering,and regulation of cell metabolism and other physiological effects.During germination and sprout growth,the phosphate group and inositol released from phytic acid degradation participates in energy metabolism and in the differentiation and development of plant cell walls,respectively,thereby regulating physiological and biochemical changes of sprouts and resisting stresses.Exogenous chemicals,such as inorganic salts and organic acids,can regulate the growth of legume sprouts,promote the degradation of phytic acid,and improve their nutritional quality.Polyamines are both a plant hormone and a signaling molecule that play an important role in plant growth and development.Ca2+acts as an important second messenger to transduce extracellular signals into intracellular physiological responses and is a major regulator of plant metabolism and growth.This study explored the mechanism of regulation of polyamines and Ca2+ on phytic acid metabolism in mung bean sprouts.The main results were as follows:1.The effects of polyamines on the phytic acid degradation and antioxidant activity of mung bean sprouts were investigated.At 72 h of germination,application of exogenous putrescine(Put),spermidine(Spd)and spermine(Spm)had a significant influence on phytic acid degradation,which exhibited concentration-dependent effects.The 18μM Spd showed the best effect.During germination,180 μM Spd treatment increased the relative expression of SPDS(spermidine syathase)and elevated the content of endogenous free,soluble conjugated and insoluble bound Spd by 104.70%,36.10%and 119.66%at 72 h of germination,which promoted the conversion of free polyamines to soluble conjugated and insoluble bound polyamines and resulted in increases in gibberellin(GA3),indole acetic acid(IAA),abscisic acid(ABA)and cytokinin(CTK).The relative expression of PA(phytase),PAP(purple acid phosphatase),MIPP(multiple inositol polyphosphate phosphatase)and ALP(alkaline phosphatase)was enhanced by Spd treatment.Then,the activities of phytase and acid phosphatase increased by 54.27%and 8.21%compared with the control at 72 h of germination,and the phytic acid content decreased by 50.13%.As a result,the inorganic phosphorus content increased significantly,energy metabolism was elevated,and the growth of the sprout、Meanwhile,the contents of total phenol and ascorbic acid were improved and antioxidant capacity was enhanced.In contrast,2 mM dicyclohexylamine(DCHA,an inhibitor of Spd synthesis)resulted in a lower SPDS expression,which reduced the content of endogenous polyamines and induced the opposite results.Exogenous Spd can reverse the inhibition of DCHA.Spd treatment significantly increased the content of water-soluble calcium,which exhibited significantly positively correlated with endogenous polyamine content and negatively correlated with phytic acid content.Thus,Spd could accelerate phytic acid degradation in mung bean sprouts by inducing the synthesis of endogenous polyamines and phytohormones and enhancing the antioxidant activities and growth of sprouts.2.The effects of CaC12 on phytic acid degradation and calcium distribution were investigated.The 6 mM CaC12 treatment reduced the expression of proton pump-related genes,increased the relative expression of Ca2+-ATPase、CaM、CDPK(calcium dependent protein kinase)and CCaMK(calcium and calcium/calmodulin-dependent serine/threonine-protein),and induced the accumulation of calcium precipitates in intercellular spaces,cell membrane,vacuole and cytoplasm.Thus,CaC12 treatment promoted Ca2+influx and transport in the cell,and increased the water-soluble calcium content,which reached a maximum at 72 h of germination,increased by 235.02%compared to the control.In addition,total calcium and CaM content were also increased.As a result,phytase activity was improved by 66.47%,and accompanied by the increment of relative expression of PA,PAP and ALP and a 44.55%in phytic acid content.The sprout growth was enhanced by CaCl2.Thus,CaCl2 could enhance Ca2+ influxes and accumulation in cells,which contributed to the regulation of phytic acid degradation.3.The effects of Ca2+ channels,DP3 and CaM on phytic acid degradation and calcium distribution were investigated.Treatment with 0.3 mM lanthanum trichloride(LaCl3,an inhibitor of plasma membrane calcium channel)caused a large accumulation of extracellular calcium precipitates,inhibited extracellular Ca2+ influx,and decreased water-soluble calcium content,resulting in opposite results.Both 0.2 mM verapamil(VP,an inhibitor of L-type calcium channel)and 0.05 mM ruthenium red(RR,an inhibitor of intracellular calcium release)changed the distribution of calcium precipitates in different compartments and resulted in the inhibition of phytic acid degradation.Among them,RR exhibited the most significant inhibitory effect.The 0.06 mM phosphatidylinositol-specific phospholipase C(PI-PLC)inhibitor neomycin(NEO)induced 86.70%reduction in Pl-PLC relative expression at 72 h of germination,resulting in 26.94%and 22.64%reduction in PI-PLC activity and(1,4,5)IP3 content,respectively.In addition,NEO caused a large accumulation of extracellular calcium precipitates,reduced water-soluble calcium and CaM content,and inhibited phytic acid degradation.Therefore,inhibiting the production of IP3 could inhibit the Ca2+ transmembrane transport,which resulted in the inhibition of phytic acid degradation.Treatment with 0.04 mM trifluoperazine(TFP,CaM antagonist)showed no significant effect on CaM content,but decreased the relative expression of proton pump and Ca2+target protein,caused a large accumulation of extracellular calcium precipitates,decreased water-soluble calcium content,and inhibited phytic acid degradation.Therefore,inhibiting Ca2+ signals and its transduction could inhibit phytic acid degradation.CaCl2 could reverse these inhibitions.4.The effects of Spd combined with CaC12 on the regulation of phytic acid degradation of mung bean sprouts were investigated.Spd treatment alone induceci the accumulation of intracellular and extracellular calcium precipitates.In the presence of Spd,the addition of CaC12 induced more calcium precipitates accumulated in the intracellular and extracellular,and the water soluble calcium and total calcium content increased significantly,which were 16.77 and 2.39 times higher than the control at 72 h of germination,respectively.Moreover,Ca+S pd treatment reduced free and soluble conjugated polyamine content,but increased insoluble bound polyamine,GA3,IAA,ABA,CTK and H2O2 content.As a result,the phytase and acid phosphatase activities were improved by 74.80%and 41.98%at 72 h of germination,respectively,and the phytic acid content was decreased by 62.27%compared to the control.Accordingly,the content of inorganic phosphorus and lower inositol phosphates under Ca+Spd treatment were all increased.The combination of Spd and CaC12 induced the lower phytic acid content than Spd and CaC12 treatment alone.Thus,Spd combined with CaCl2 could promote the degradation of polyamines and increase hormone levels by promoting the absorption and transport of polyamines and Ca2+,thereby promoting phytic acid degradation,increasing energy metabolism,and promoting the elongation of embryonic axis cells and the growth of green bean sprouts.Spd combined with CaCl treatment had a synergy effect on phytic acid degradation.5.The connection of Spd and Ca2+in phytic acid degradation of mung bean sprouts was studied.DCHA treatment alone induced many calcium precipitates accumulated in cell wall and cell membrane.In the presence of DCHA,the supplementation of CaC12 enhanced the accumulation of extracellular calcium precipitates,and elevated the water-soluble calcium and total calcium contents,which were 15.54 and 2.15 times higher than the control at 72 hours of gernination,respectively.Moreover,Ca+ DCHA treatment promoted the conversion of free polyamines to insoluble bound polyamines and increased the content of H2O2.In addition,Ca+ DCHA addition induced the increase in hormone contents and the relative expression of PAP and ALP,and increased the activity of phytase and acid phosphatase by 29.80%and 31.69%,respectively,compared with the control at 72 h of germination.As a result,phytate content was decreased by 29.25%.Therefore,the addition of CaCl2 reversed the DCHA inhibition of phytic acid degradation,and promoted cell elongation and growth.In the presence of Ca+DCHA,the addition of exogenous Spd had no significant effect on the degradation of phytic acid.Exogenous Spd could reverse the inhibitory effect of La on Ca2+ influx·There was no macroscopic calcium precipitates in cell compartments under Spd+La treatment.No significant difference in water-soluble calcium contents were detected between control and Spd+ La-treated sprotus.However,Spd+La treatment caused large accumulations of endogenous free,soluble conjugated and insoluble bound polyamines.And the free,soluble conjugated and insoluble bound Spd increased by 389.70%,281.37%,and 289.85%,respectively,compared with the control at 72 h of germination.Conversely,H2O2 content under Spd+La treatment was significantly reduced,thus polyamine degradation was inhibited,and GA3,ABA,and CTK levels were decreased.As a result,the phytic acid content was increased by 19.35%compared with the control,and the length of the hypocotyl cells was significantly smaller than the control,and the inhibitory efFect of LaC13 on phytic acid degradation and the growth of the sprouts could not be reversed by Spd.Therefore,Ca2+ acted as a downstream signal of Spd in the regulation of phytic acid degradation of mung bean sprouts.6.Metabolic profiles of of mung bean sprouts under Spd and CaCl2 were investigated.In the metabolomic analysis of mung bean sprouts treated with CaC12,Spd,and CaCl2-Spd,a total of 102 and 87 differential-expressed metabolites were identified in pos and neg mode,respectively.These differential metabolites indicated their connection with amino acid biosynthesis and metabolism,phenolic compounds biosynthesis,terpenoids biosynthesis and degradation,fatty acid metabolism,ABC transporters,folate biosynthesis,carbohydrates metabolism,phosphatidylinositol signaling system,biosynthesis of plant hormones,indole alkaloid biosynthesis,nucleotide metabolism,and Ubiquinone and other terpenoid-quinone biosynthesis.Among them,the down-regulated metabolites indicated their connection with carbohydrates metabolism,biosynthesis and degradation of amino acids,phenolic compounds biosynthesis,linoleic acid metabolism,purine metabolism and ABC transporters,while the up-regulated metabolites indicated their connection with biosynthesis of plant hormones,amino acid metabolism,linolenic acid metabolism,indole alkaloid biosynthesis and biosynthesis and degradation of terpenoids.Among them,CaCl2-Spd promoted phytic acid degradation resulting from the enhancement of inositol phosphate metabolism,tricarboxylic acid cycle and phytohormone biosynthesis.