Physiological Characteristics Of Response To Magnesium Stress In Mulberry And Identification Of Functional Genes Of MmNIA

Mg²⁺is the most abundant divalent cation in plant cells and an important component of chlorophyll,which is involved in photosynthesis and various physiological metabolic processes of plants,and plays an important role in plant growth and development.Due to the influence of geological factors,magnesium deficiency or magnesium toxicity may occur in soil.The mechanism of how plants respond to magnesium stress is poorly understood.In this study,“Yu 71-1”was selected as the test material,3 m M Mg SO₄（CK）was used as the control,and five concentration gradients were set:0 m M Mg SO₄（T1）,1 m M Mg SO₄（T2）,2 m M Mg SO₄（T3）in the magnesium deficiency group,and 6 m M Mg SO₄（T5）,9 m M Mg SO₄（T6）in the high magnesium group.Three biological replicates were set in each treatment group for20 days.Leaves with the same leaf position and the same growth potential were collected to study the growth and development of mulberry seedlings,in vivo nutrition,physiological and biochemical indexes,photosynthetic characteristics,transcriptome and other aspects.Through transcriptome sequencing data analysis,the nitrate reductase gene MmNIA,a key response gene to magnesium stress,was cloned and its function was identified by subcellular localization and VIGS silencing techniques.The response mechanism of mulberry to magnesium stress was revealed from physiological and molecular aspects.The results provided some molecular basis for mining key genes of magnesium tolerance and breeding new varieties of mulberry.The main research results are as follows:1.Structural stability and photosynthetic characteristics of mulberry under magnesium stress:Complete and stable structure is the basis of plant photosynthesis,and a certain amount of magnesium supply is conducive to the stability of mulberry leaf structure and the improvement of photosynthetic efficiency.Experimental studies have shown that:（1）When treated with 3 m M Mg SO₄,Mulberry had excellent growth and regular chloroplast structure,while when treated with low magnesium（T1、T2）,the chloroplast and mitochondrial structure were deformed and disintegrated,the membrane structure was damaged,and the number of chloroplast grana and lamella were significantly reduced.（2）Compared with CK,net photosynthesis（A）,transpiration rate（E）and stomatal conductance（Gs）were significantly decreased under magnesium deficiency（T1～T3）and high magnesium（T5,T6）treatments.（3）Chlorophyll a（Chla）,chlorophyll b（Chlb）and chlorophyll（chl）were higher than those in other magnesium treatment groups at 3 m M Mg SO₄.2.Analysis on the activities of osmotic regulatory substances and protective enzymes in mulberry leaves under magnesium stress:The activities of osmotic regulatory substances and protective enzymes in mulberry leaves were analyzed.The results showed that superoxide dismutase（SOD）,peroxidase（POD）and catalase（CAT）of 3 m M Mg SO4 were significantly increased compared with T1 and T6groups（P<0.05）,and the content of reactive oxygen species（ROS）was kept at a low level.Compared with CK group,the contents of soluble protein（SP）and soluble sugar（SS）in mulberry leaves were significantly decreased（P<0.05）under magnesium deficiency stress（T1～T3）and high magnesium stress（T5～T6）,and the synthesis of these two substances was blocked under stress.Compared with CK,the contents of malondialdehyde（MDA）and proline（PRO）in T1 and T6 groups were significantly increased（P<0.05）,which could reduce water loss and maintain osmotic balance of cells.3.Analysis of nutrient element balance in plants under magnesium stress:Nutrient elements are the basis of plant growth and metabolism,and appropriate magnesium concentration is conducive to plant growth and development and normal life activities.The results showed as follows:（1）Magnesium content in roots,stems and leaves of mulberry seedlings under low magnesium concentration（0～2 m M Mg SO4）treatment showed a decreasing trend compared with CK（3 m M Mg SO4）,and magnesium content in all organs showed a decreasing trend when magnesium supply was excessive.（2）With the increase of Mg²⁺concentration,the contents of N,P,K,Ca,Fe,Zn,Cu and S in all organs of mulberry showed no obvious rule,while Mg showed a trend of increasing first and then decreasing.The content of magnesium in mulberry leaves was positively correlated with the concentration of magnesium treatment,nitrogen content（P<0.01）,potassium content（P<0.01）and calcium content.It showed synergistic effect,but it was negatively correlated with phosphorus content,showing antagonistic effect.（3）The basic distribution rules of nutrient elements were:（N,K）leaf>stem>root;（Mg）root>stem>leaf;（Fe）root>leaf>stem;（Zn）stem>leaf>root.4.Transcriptome sequencing analysis of Mulberry response to magnesium stress:Based on the changes of morphological structure,photosynthetic characteristics,physiological and biochemical indexes of mulberry under magnesium stress,the transcriptome sequencing of mulberry leaf samples treated with different concentrations of magnesium showed that the average output of 6 Gb data of mulberry leaf samples with different concentrations of magnesium,27,085 genes could be detected,28,489 were known genes,and 1404 were predicted new genes.The number of differential genes in CK-vs-T1,CK-vs-T2,CK-vs-T3,CK-vs-T5 and CK-vs-T6 groups was 2276,3380,1569,1061 and 2802,respectively,among which 377 genes were shared among the five groups.The results of q RT-PCR showed that the transcriptome sequencing data were accurate and reliable.The GO annotation results showed that differential genes were mainly annotated in metabolic processes,cellular processes and single tissue processes in biological processes,cell components in cells,organelles and membranes,and catalytic,binding and other functions in molecular functions.KEGG results showed that the differential genes were mainly concentrated in the flavonoid biosynthetic pathway,sesquiterpene and triterpene biosynthetic pathway,and the biosynthetic pathway of secondary metabolites.5.Cloning,expression,localization and functional identification of MmNIA gene in Mulberry:According to the transcriptional sequencing results of mulberry magnesium stress,NIA gene was the most significantly expressed among the differentially expressed genes of nitrogen metabolism pathway.Mulberry MmNIA gene was cloned,the CDS sequence was2730bp,encoding 909 amino acids,predicted protein 102.3KDa,isoelectric point 6.97.The predicted molecular weight of the protein is 102.3Kda,and the theoretical isoelectric point is6.76.Phylogenetic tree was used to analyze the homology and genetic relationship with other plant species,and the results showed that it was close to mulberry,mulberry and hemp.The expression level of MmNIA gene in mulberry leaves under magnesium stress was detected by q RT-PCR,The expression level in groups T1,T2 and T5 was significantly higher than that in CK group.The protoplast subcellular mapping technique was used to determine that the gene might be located in the cytoplasm.The VIGS technique was used to silence the target fragment of MmNIA gene.The expression level of MmNIA in mulberry leaf samples,the activity of nitrate reductase in leaves and the content of magnesium in leaves in experimental group were significantly decreased compared with blank control group and empty carrier control group,and the photosynthetic capacity was decreased.It is speculated that the gene MmNIA influences the photosynthetic capacity of mulberry by regulating the expression of nitrate reductase through the nitrogen metabolism pathway of mulberry.Therefore,the gene MmNIA can provide certain molecular basis for the response of mulberry to magnesium stress and the cultivation of magnesia-tolerant mulberry varieties.