Mechanisms Of Lignin Accumulation In The Lignified Leaf Veins Of The Magnesium Deficient Citrus Sinensis Seedlings

Citrus is not only the largest fruit in the world,but also the largest fruit in China.In China,citrus is cultivated in the Yangtze River Basin and the subtropical area to its south.With the continuous increase of citrus yield and planting area in China,the insufficient supply of soil nutrients and the imbalance of nutrient supply have become important factors affecting the sustainable development of citrus industry,especially the deficiency of Mg in soils.Similar to the Mg-deficiency of citrus,Mg-deficiency may lead to leaf interveinal chlorosis,as well as lignification,swelling and cracking of veins in citrus leaves.Perennial Mg-deficiency in citrus orchards is easy to cause tree nutrition imbalance and reduce fruit yield.So far,there are few studies on the mechanism of vein lignification caused by Mg-deficiency in citrus leaves.The seedlings of sand cultured‘Xuegan’[Citrus sinensis（L.）Osbeck]were treated with0 m M（Mg-deficiency）or 2 m M（Mg sufficiency）Mg（NO₃）₂ for 16weeks.Thereafter,the effects of Mg-deficiency on the photosynthetic characteristics of leaves and the nutrient contents of leaves and veins were investigated.Also,the molecular physiological mechanisms of lignin accumulation in leaf veins of Mg-deficiency were investigated based on physiology,transcriptome,proteome and metabolome.The main results are as follows:1.Mg-deficiency inhibited CO₂ assimilation and induced vein lignification in middle and lower leavesLeaf interveinal chlorosis,vein lignification,swelling and cracking,as well as the changes of photosynthesis and related parameters induced by Mg-deficiency were greater in the lower leaves than in the middle leaves,but these phenomena were not observed in the upper leaves.This might be related to the increased transport of Mg from old leaves to young leaves under Mg-deficiency,because Mg concentration in leaves decreased with the increase of leaf age.The lignification,swelling and cracking of leaf veins caused by Mg-deficiency were not caused by boron-deficiency due to Mg-deficiency.The decrease of CO₂ assimilation rate in middle and lower leaves under Mg-deficiency was related to the decrease of energy absorption and transfer efficiency caused by Mg-deficiency-induced impairment of photosynthetic electron transport chain.2.Proteomic analysis indicated that Mg-deficiency-induced vein lignification was related to the increase of protein abundance involved in phenylpropane biosynthesis,hormone biosynthesis and transportation,and glutathione metabolismThe concentrations of total soluble sugar（glucose+fructose+sucrose）,starch and cellulose in Mg-deficiency veins of lower leaves（MDVLL）were less than those in Mg-sufficiency veins of lower leaves（MSVLL）,but the concentration of lignin was higher than that in MSVLL.However,there was no significant difference in these four parameters between Mg-deficiency veins of upper leaves（MDVUL）and Mg-sufficiency veins of upper leaves（MSVLL）.Mg-deficiency-induced alterations of non-structural carbohydrates,cell wall components and protein contents in the veins of lower leaves than those in the veins of upper leaves.Using label-free,liquid chromatography-tandem mass spectrometry（LC-MS/MS）analysis,1229 and 492 differentially abundant proteins（DAPs）were identified from MDVLL vs MSVLL and MDVUL vs MSVUL,respectively.Further analysis showed that in MDVLL vs MSVLL,vein swelling and cracking caused by the increase of lignin concentration might be related to（1）increased abundances of proteins involved in phenylpropane biosynthesis pathway,hormone biosynthesis and glutathione metabolism;（2）increased abundances of other DAPs（such as Cu/Zn SOD,Ao and ABC transporters）involved in lignin biosynthesis;and（3）increased abundance of proteins[such as expansins,Rho GTPase activating protein gac A,ascorbic acid oxidase（AO）,monocopper oxidation like protein and xylan endoglycosyltrans-ferases（XTHs）]involved in cell wall metabolism.3.Transcriptome analysis showed that Mg-deficiency-induced vein lignification was related to the upregulation of transcription factors and genes involved in lignin biosynthesis,cell cycle regulation and cell wall metabolismUsing RNA-Seq,3065 upregulated and 1220 downregulated genes and 1390 upregulated and 375 downregulated genes were obtained from MDVLL vs MSVLL and MDVUL vs MSVUL,respectively.There were1473 common differentially expressed genes（DEGs）in MDVLL vs MSVLL and MDVUL vs MSVUL,of which 1463 DEGs had the same expression trend.Further analysis indicated that Mg-deficiency-induced lignification,swelling and cracking in the veins of lower leaves were related to（1）the upregulation of many transcription factors and genes involved in lignin biosynthesis,cell cycle regulation and cell wall metabolism;and（2）the activation of reactive oxygen species（ROS）,plant hormones and cell wall integrity（CWI）signals.4.Metabolomic analysis showed that Mg-deficiency-induced vein lignification was related to the increase of precursor metabolites involved in lignin synthesisUsing the widely targeted metabolomics based on UPLC-MS/MS,a total of 344 differentially abundant metabolites（DAMs）were detected from MDVLL vs MSVLL,of which 200 were upregulated and 144 were down regulated.Further analysis showed that（1）Mg deficiency promoted the accumulation of precursors sugars（D-glucose-6-phosphate and glucose-1-phosphate）and amino acids（phenylalanine,tryptophan,proline and ornithine）in leaf veins;（2）Mg-deficiency up regulated phenylpropane biosynthesis and inhibited flavonoid synthesis,so as to promoted the synthesis of typical lignin monomers（p-coumarinol,mustard alcohol and coniferol）in leaf veins.（3）Mg-deficiency promoted the accumulation of lignin monomers phenolic amines（ferulic tyramine）in leaf veins.In conclusion,in MDVLL vs MSVLL,the concentrations of a large number of metabolites related to lignin synthesis（sugars,amino acids,phenylpropanoids and phenolic amines）increased significantly,which promoted the accumulation of lignin.Combined multiomics analysis showed that sucrose metabolism,pentose phosphate pathway,glycolysis pathway,arginine proline metabolism,flavone flavonol synthesis,phenylalanine metabolism and phenolic amine metabolism may be involved in Mg-deficiency-induced lignification,expansion and cracking of leaf veins.In conclusion,this present study revealed the accumulation mechanisms of lignin in lignified leaf veins of C.sinensis from the aspects of physiology,transcriptome,proteome and metabolome,provided a theoretical basis for further revealing the adaptation mechanisms of citrus to Mg-deficiency,and provided a scientific basis for finally improving the Mg-deficiency tolerance of citrus and realizing high-yield and high-quality cultivation of citrus.