Fruit Calcium Uptake Patterns,Dynamics Of Calcium Oxalate Content In The Pedicles Of Litchi And Longan Fruits And Their Relationship To Germin-like Proteins

Calcium plays an important role in the formation and maintenance of the fruit quality and its deficiency causes various physiological diseases.It was found that the calcium concentrationin fruit stalk was significantly higher than in the fruit of litchi and that there might be a bottleneck of calcium transport to the fruit in the pedicel.High calcium can besequestered by forming calcium oxalate whichmakes calcium immobile.Recent reports showed that the germin-like proteins(GLPs)indentified in dicots decomposes oxalic acid and releases calcium ions.Is the bottleneck of calcium transport in litchi and longan pedicels related to the formation of calcium oxalate?Can calcium oxalate accumulated be degraded again and calcium sequestered be reutilized? These questions are yet to be answered.Although there have been intensive studies about the formation of calcium oxalate,its functions are not well known.In this study,crack-resistant litchi cv‘Lingfengnuo’ and longan cv.’Shixia’ and cracking-susceptible litchi cv.’Nuomici’ were used as the experimental materialsto analyze their differences infruit calcium uptake,dynamics of various forms of calcium,oxalic acid content and oxalate oxidase activity,and their correlations.GLPs were cloned from litchi and longan and their expression patterns were analyzed in relation to oxalic acid and calcium oxalate.The purpose of the study is to obtain understandings of the physiological mechanism of calcium uptake in fruit.Major results are below:1.Cracking-susceptible ’Nuomici’ litchi had a lower calcium uptake capacity than cracking-resistant‘Shixia’’ longan and‘Lingfengnuo’ litchi.In the three materials,calcium concentrations in the pedicelwere always higher than those in the fruit throughout fruit development.Fruit calcium content was significantly lower than’Lingfengnuo’ and ’Shixia’during rapid aril growth(ca 60 days after anthesis),when peak of fruit cracking occurs.2.Calcium oxalate crystals are present in the pedicel.Electron probe analysis revealed calcium rich zones in the pith and phloem and higher calcium level in the phloem than the xylem,suggesting that phloem might also be serve as the pathway of calcium transport to fruit.Under optical microscope,phloem cells were shown to contain calcium oxalate crystals,which were chiefly in diamond shape.3.During fruit development,calcium oxalate accumulated in the pedicel could be dissolved.Soluble calcium,structural calcium and oxalate calcium changed dynamically,suggesting that calcium oxalate crystals can be dissolved.Calcium oxalate in the pedicels in litchi and longan was generally higher in the early stages than in the later stages,and might serves as temporary calcium pool,which was released during rapid aril growth for structural need.’Nuomici’ pedicel had the lowest calcium oxalate content in the early stages,but in the late stages calcium oxalate in ’Nuomici’ pedicel accumulated while it decreased in’Lingfengnuo’ and ’Shixia’.Therefore,the ’Nuomici’ had the lowest capacity in reutilization of calcium in oxalate form.4.Correlations exited between oxalate oxidase activity and contents of oxalic acid and calcium oxalate.In this study,we first detected activity of oxalate oxidase.The enzyme activity was highest in the early stages,decreased to a lowest value around 30 days after anthesis and maintained low thereafter.Oxalic acid content displayed negative correlation with oxalate oxidase activity in both the pedicel and the pericarp in all tested materials.In the pedicel,calcium oxalate content showed a negative correlation with the enzyme activity.However,in the pericarp,a significant correlation was found between calcium oxalate and the enzyme activity.The results suggested that in pedicel oxalate oxidase participated the metabolism of oxalic acid and calcium oxalate and played an important role in releasing calcium from calcium oxalate.The enzyme activity was weakest in ’Nuomici’ with the lowest capacity in calcium uptake,which might be related to its weakest capacity in calcium release from calcium oxalate crystals and thus continuous calcium oxalate accumulation in the late stages.5.Genes encoding GLPs were cloned from litchi and longan and their expressions were analyzed in relation to oxalate oxidase.Five GLP genes were separately cloned from litchi(LcGLP1-5)and longan(DlGLP1-5).Bioinformatic analysis showed that their encoded proteins contained RDG domain and conserved Box A and Box B,corresponding to the conserved active site.LcGLP1,LcGLP2 and LcGLP3 were found to contain premature translation termination condons and their expression levels were very low.The expression of the other GLPs were tissue-specific,all being more highly expressed in the pericarp and pedicel.The expression levels of DlGLP1,DlGLP3 and Dl GLP5 in longan pedicel were significantly and negatively correlated to oxalate oxidase activity,while in’Lingfengnuo’ litchi the expression levels of LcGLP4 and LcGLP5 had significant positivecorrelation with the enzyme activity.Yet in ’Nuomici’,only LcGLP4 was positively correlated to enzyme activity.The results suggested that LcGLP4 might be the gene encoding oxalate oxidase in litchi.