Impact Of Calcium Stress And Root Restriction On Development And Metabolites Of Grape (Vitis Vinifera L.)

Among the essential elements required for plant growth and development,calcium plays a vital role in cell structure and physiological function.It participates in the regulation of plant growth,development and metabolism.After absorption from roots,it can only be transported via xylem system.Low mobility of calcium causes the disturbed absorption and distribution,which may restrict a number of physiological functions in plant.Unfortunately,Calcium absorption can be inhibited by low soil fertility and environmental deterioration;subsequently,its deficiency can leads to abnormal growth,development and physiological disorders.Root-zone restriction is a new type of fruit cultivation technique,which can modify the quality of root soil and promote the absorption and transport of calcium.In this study,grapevine(Vitis vinifera L.)was selected as a testing material.The effects of calcium deficiency on the growth and photosynthesis of grape seedlings were analyzed by plant tissue anatomy and physiological methods.The metabolomics analysis were used to study the changes between primary and secondary metabolites of grape berry in accordance to calcium deficiency and root-zone restriction treatment.The goal of this research was to clarify the mechanism of calcium deficiency on the growth and metabolism in grapes;thereafter,the pathway for promoting calcium absorption and improving grape cultivation technique will be discussed as well.The main results are as follows:1.One year old seedlings of Thompson seedless grapevines were cultured with-Ca(Ca2+:0 mM).After 24 days of treatment(DAT),the leaves showed typical calcium deficiency symptoms with yellowing between veins.After 52 DAT,the edges of the leaves began to wither and curl downward,accompanied by growth cease and necrosis of shoot apex tip.The reduction of CO2 assimilation capacity with-Ca was due to decreased function of photosynthetic apparatus,especially the integrated limitations of biochemical processes(Jmax and VCmax).2.The growth and vascular structure of grape seedlings cultured for 52 days under-Ca were studied.It was found that the vegetative growth of grape seedlings was inhibited,the diameter and area of vessel cells decreased,the number increased,and the cell wall thickened.At the same time,we also found that-Ca affected the differentiation of lateral roots and changed the calcium oxalate crystals in parenchyma cells.Ultrastructural studies of stem showed that-Ca resulted in the plasmolysis,cytosol leakage and organelles broken of cambium and phloem cell.Analysis of mineral elements in different organs of plants under-Ca showed that calcium deficiency significantly reduced the concentrations of N,Ca,Fe and Cu in plants,while it increased the concentrations of Mg in shoot and leaves,and B in roots and leaves.3.Red Alexander grapes cultured with different calcium concentrations(Ca2+:0 mM;0.2 mM;4 mM)were used to study the effects of different calcium concentration on the vegetative growth and the changes of soluble sugar and organic acid content in different tissues of grapevines.The results showed that the grapevines vegetative growth were inhibited by-Ca and LCa,however,the phenological stage were advanced than that of+Ca.The skin,pulp,seed and leaf employed different strategies of sugar and organic acid metabolism in response to calcium stress,which showed tissues specificity.The skin is more sensitive to calcium concentration because of largest changes with sugar and acid;the seeds requirement could be met by low calcium;and the leaf showed a relatively earlier response to calcium stress.4.Four-year old Thompson seedless grapes were treated by three calcium concentration(-Ca:0 mM;+Ca:4 mM;HCa:8 mM)to study the variation of metabolomic of ripening grape berries.Non-targeted metabonomics was employed in this experiment.124 metabolites were successfully identified in grape fruits,and the metabolites shifts in ripening fruits under-Ca,+Ca and HCa culture conditions were compared.Compared with+Ca,the effects of-Ca and HCa on fruit metabolism at ripening focused on the changes of amino acids,which were located in glutamate pathway,aspartic acid pathway and aromatic amino acid pathway,respectively.The metabolites of amino acids in glutamate pathway and aspartic acid pathway decreased under-Ca,and increased under HCa,but both glutamate and aspartic acid and their precursor citric acid increased under two stresses.Aromatic amino acids are the precursors of secondary metabolites synthesis.Aromatic amino acids are precursors for the synthesis of secondary metabolites,their content is reduced under-Ca and HCa stress,so most of the metabolites in the secondary metabolic pathway are decreased,including flavanols and flavonols,etc.This study extends our understanding of shifts in grape fruit metabolites under calcium stress and provides a reference for practical production.5.Four-year-old Red Alexander grapes were used as materials,291 metabolites were identified by non-targeted metabolomics methods in the developing skin and pulp tissues under root-zone restriction and traditional cultivation(control),respectively.Shifts in metabolomics of the developmental skin and pulp under root restriction and control conditions were analysed.Principal component analysis showed that the metabolism of the skin and pulp was mainly affected by the fruit development stage,followed by the cultivation method.Compared with the control,root restriction decreased the content of amino acids accompanied with coloring advanced,except the key stress-responsive amino acids(e.g.2-aminohexanedioic acid,aspartic acid,glutamine and serine).As precursors of secondary metabolites,the increased content of aromatic amino acids(phenylalanine,tryptophan and tyramine)led to the synthesis of secondary metabolites increased significantly,which providing a direct evidence for the transition from primary metabolism to secondary metabolism earlier and faster.