Effect Of Light On The Formation Of Calcium Oxalate Crystals In The Cells Of Mammillaria Schumannii And Pitaya

Biomineralization is the process by which organic life controls the production of inorganic minerals and is a frequently occurring phenomenon in nature.The formation of calcium oxalate crystals in crystal-idoblast is a basic and universal physiological metabolic process.In this study,the morphology and distribution of calcium oxalate crystals in tubercles of Mammillaria schumannii were observed by strong light and weak light treatments,the concentrations of total oxalic acid and calcium ions,and stomatal related indexes in tubercles were determined,and then their correlation with photosynthesis was analyzed.Following a discussion of the effects of various isolation techniques on the isolation quantity and crystallization of calcium oxalate,and followed by combining PLM,SEM,EDS and Raman spectroscopy.The morphology and structure of calcium oxalate crystals in plants and isolated calcium oxalate crystals were observed and analyzed,as well as the organic matter wrapped by spherical druse crystals.The main results are as follows:1.Effect of weak light on the cell structure of tubercles in Mammillaria schumanniiThe tubercles changed from dark green to tender green after weak light treatment,the stems elongated and grew,the distance between tubercles broadened,the tubercles became slender,and the thorns softened slightly.Compared with the strong light treatment,the epidermal cover became thinner,the cortical cells became thicker,and the stomata enlarged with the prolongation of the weak light treatment time（5,10,and 15 days）;the palisade and spongy parenchyma cells became larger and more rounded,and the number of chloroplasts decreased;the arrangement of xylem and phloem in vascular bundles became loose,and the pith cells in the middle became larger.Obviously,plants respond to weak light stress by changing the size of their cells.The Raman characterization analysis of the epidermis showed that the epidermic covering became thinner gradually from strong light to weak light.The stomatal opening and density of epidermis were observed under SEM,it was found that the density increased and then decreased with the increase of weak light treatment time,and the stomatal opening increased gradually,and the stomatal opening under weak light treatment had a very significant difference（p<0.001）.Expose to strong light,low stomatal density and small opening are beneficial to water retention;after weak light treatment,the increase of stomatal density and opening is more conducive to photosynthesis.EDS showed that the content of K⁺in the epidermis increased after weak light treatment,indicating that weak light stress led to the increase of K⁺content,resulting in the decrease of water potential,cell water absorption,stomatal opening,and promote photosynthesis.2.Distribution characteristics of calcium oxalate crystals in tuberclesSmall-sized calcium oxalate crystals are mainly distributed in the palisade crystalline cells of the epidermis and cortex;spongy parenchyma cells near the vascular bundles,a few cells will increase the specificity to form large-size calcium oxalate crystals.Compared with the crystal density in warts exposed to strong light（weak light treatment for 0 days）,the crystal density of calcium oxalate increased after weak light treatment,and the difference was very significant（P<0.001）.Compared with the crystal size in warts exposed to strong light,the crystal size decreased after weak light treatment,and the difference was very significant（P<0.001）.The contents of total oxalic acid and calcium in plants were determined.The results showed that the concentration of total oxalic acid was 1.1006 mg/m L under strong light and 0.7692 mg/m L under low light treatment.The extracted solution containing Ca²⁺was measured by electrically coupled plasma emission spectrometer.The results showed that the content of Ca²⁺was 102.4577 mg/L under strong light treatment and 122.1436 mg/L under low light treatment.3.Isolation of calcium oxalate crystals from tuberclesMechanical crushing after liquid nitrogen freezing,adding distilled water and isopropanol during crushing,and mechanical crushing are not ideal methods for crystal extraction.In this study,the method of differential centrifugation after mechanical crushing can be used to separate not only large-size calcium oxalate crystals,but also 70%small calcium oxalate crystals with a size of less than 20μm.4.Physicochemical characterization of mineral crystals in tuberclesThere are mineral crystals of calcium oxalate,calcium phosphate,and silica in Mammillaria schumanni,and calcium oxalate crystals are dominant.They can be divided into two morphologies:prisms and clusters,the clusters include spherical spherical durses and prismatic clusters under strong light and prismatic（tetrahedron,octahedron,prism aggregates）crystals under weak light.The analysis by Scanning Electron Microscope and X-ray Energy Spectrum showed that the crystals were mainly Ca C₂O₄·2H₂O.In addition,we found that spherical druses had 2 obvious abnormal peaks of-CH and-OH at 2,947 and 3,290 cm^-1,which may be attributed to the existing organic matrix.Raman analysis of the organic matrix further extracted from the crystals indicated that polysaccharide,flavone,or lipid compounds on the basis of bands at 2,650,2,720,2,770,and 2,958 cm^-1.5.Observation of calcium oxalate crystals in the succulent stem of dragon fruitIt was observed by SEM that calcium oxalate in the succulent stem of dragon fruit treated with weak light were prism and distributed in the epidermis（small-sized）and parenchyma cells near the vascular bundle（large-sized）.Although durses are also formed in the succulent stems treated with weak light,there are no spherical druses,which is consistent with the founding in the Mammillaria schumanni.Furthermore,it is demonstrated that when treated with weak light,the morphology of crystals changes from spherical durse to prism.We speculate that there seems to be some organic matrix regulating the morphology of calcium oxalate crystals,and the morphology and distribution of calcium oxalate crystals are closely related to their function.