Effects Of Coumarin And Caffeic Acid On Morphological And Structure Of Young Roots Of Alfalfa And Rotation Crops And Their Physiological Changes

As a high-quality perennial leguminous forage,Alfalfa has the characteristics of one-time planting,multi-year harvest,high grass yield,rich nutrition and good palatability,etc.It is an important source of high-quality protein feed and is called"king of forage".But because alfalfa is autotoxic,it is difficult to grow alfalfa soil again.At present,the mechanism of Allelopathy action on themselves,wheat and corn from higher content of coumarin and caffeic acid in alfalfa have rarely reported.In particular,the effects on the anatomical structure,the endogenous hormones and the molecular mechanism of young alfalfa,wheat and cornroots had not systematically reported.In this study,the petri dish culture method was used to test the exogenous coumarin,caffeic acid and their mixture in individual alfalfa,wheat and corn seeds,and the germination rate,germination index,main root length,seedling height,root tip growth,root cell viability,root pectin methylesterase?PME?and allelopathic effect index were compared of the three crops.An experiment of the nutrient solution sand culture was used to add exogenous coumarin,caffeic acid and their mixture which concentration gradient was 0,0.5,5,50,500 mg·L^-11 on alfalfa,wheat and corn.Paraffin Sectioning Technique and the root scanning system were used to compare the effects of exogenous additives on the root morphological parameters such as total root length,total root surface area,root volume,and root tip number and on root anatomical indexes such as root thickness,cortical thickness,diameter and development of vascular cylinder,number and area of xylem vessels.The content dynamics and effect characteristics of ABA,GA₃,IAA and ZT in roots of three kinds of seedlings were detected and analyzed by HPLC.High performance liquid chromatography and real-time fluorescent quantitative PCR were used to quantitatively analyze the regulation of ABA content and ABA synthesis key enzymes NCED,ZEP and BG in the roots of alfalfa,wheat and corn seedlings by coumarin and caffeic acid.And explore the relationship between crop types,ABA content and synthetic key enzyme genes,and the following main results were obtained.1.The allelopathic inhibitory effect of coumarin,caffeic acid and coumarin+caffeic acid mixture on wheat seed germination from strong to weak was coumarin>mixture>caffeic acid,and the inhibition effect on corn seed was as mixture>coumarin,Caffeic acid showed promote allelopathy.With the increase of additive concentration,wheat and corn seed germination and seedling growth inhibition intensity increased,but the inhibition degree of wheat with the same concentration and the same kind of additive was stronger than that of corn.2.Both caffeic acid and coumarin+caffeic acid mixture took 50 mg·L^-11 as the inflection point of positive and negative effects,and coumarin took 5 mg·L^-11 as the inflection point of positive and negative effects,with the increase of the concentration of exogenous additives,the promoting effect on the root elongation of alfalfa was gradually weakened.Coumarin and caffeic acid of 5 mg·L^-1significantly promoted the elongation of maize roots.500 mg·L^-11 of coumarin and coumarin+caffeic acid mixture showed significant elongation to wheat and corn roots,and the inhibitory effect of coumarin+caffeic acid mixture was stronger than that of coumarin.The concentration threshold of alfalfa root border cells to resist coumarin,caffeic acid and coumarin+caffeic acid mixture stress was 5 mg·L^-1,500 mg·L^-1,50 mg·L^-1,respectively.The concentration threshold of wheat and corn root border cells to resist coumarin and coumarin+caffeic acid mixture stress respectively was 500 mg·L^-1,50mg·L^-1and 50 mg·L^-1,500 mg·L^-1.3.The coumarin+caffeic acid mixture treatment with 0.5 mg·L^-1showed a significant promotion effect on the development of anatomical structures such as cortex,middle column and catheter of alfalfa young roots,and enhanced the promotion effect of monomeric coumarin and caffeic acid?P<0.05?.The allelopathic effects of exogenous additives on wheat and corn seedlings were promoted by low concentration and inhibited by500 mg·L^-1,When the additives were treated at low concentration,the allelopathic effect on wheat were from strong to weak in the order of caffeic acid>coumarin+caffeic acid mixture>coumarin,and caffeic acid had the strongest promoting effect on corn.When the additive was treated at a high concentration,the allelopathic inhibition effect on wheat were from strong to weakin the order of coumarin+caffeic acid mixture>coumarin>caffeic acid,and the coumarin+caffeic acid mixture was most effective in suppressing corn.Compared with wheat,the allelopathic comprehensive inhibition effect of three exogenous additives on cron root morphogenesis were weaker than that of wheat under high concentration treatment with the extension of treatment time.4.The inhibitory effect of caffeic acid on the synthesis of IAA,ZT and GA₃ which were endogenous hormones in root system of three crops was the weakest,and the promoting effect of ABA was also the weakest in the range of threshold(50 mg·L^-1).the hormone ABA has played an important role in the growth and development of alfalfa,wheat and corn under the stress of coumarin and caffeic acid.The change trend of endogenous hormones IAA and ZT were basically consistent in the roots of cron seedlings.and there was a significant positive correlation between the ratios of hormones.5.The coumarin+caffeic acid mixture treatment which stimulated the relative expression of key enzymes NCED,ZEP and BG in the ABA synthesis pathway to a certain extent,and increased the ABA content in alfalfa roots and enhanced the autotoxicity of alfalfa against coumarin and caffeic acid.The relative expression levels of NCED and ZEP in alfalfa were higher than those in wheat and corn,but the relative expression of BG in alfalfa was lower than that in wheat and cron.The linear correlation between the relative expression of NCED and ZEP and ABA content was similar to that of alfalfa and wheat,while the relative expression of ZEP and ABA content was extremely significant in corn?P<0.01?.The effects of exogenous additives on ABA content and related synthetic gene expression of alfalfa were the most obvious,and corn was the weakest.In the process of ABA synthesis,the gene of NCED plays a major role in the action of coumarin,while the gene of NCED and ZEP both play a positive role in the action of caffeic acid and compounds.