The Study Of Fruit Calcium Uptake Pattern,Pathways And Regulatory Mechanisms

Calcium is one of the essential elements for fruit trees.Deficiency of calcium in fruit causes physiological disorders leading to quality loss.Role of phloem pathway in calcium transport into fruit is still a matter of dispute.A clear understanding of the pathways and influencing factors is crucial to understanding how calcium uptake in fruit is regulated,which is important to tackle Ca-related disorders.In this experiment,different species and varieties of fruits were used as experimental materials to study fruit calcium uptake pattern and differences in fruit uptake capacity among species/varieties,to analyze influencing factors and the common relations to fruit calcium uptake and the role pathways of phloem and xylem in fruit calcium uptake.On the top of that,a case study in litchi was carried out to reveal the role of various calcium transporter genes in fruit calcium uptake in order to understand the molecular physiological mechanisms of fruit calcium uptake.The main results of the study are as follows:(1)The dynamic patterns of fruit growth and calcium uptake in 11 varieties of 8 fruit species were analyzed.Two indexes,fruit calcium uptake rate and fruit calcium uptake activity were used to reflect fruit calcium uptake capacity.The result showed that fruit calcium capacity differed significantly among species/varieties,and the differences among species were greater than among varieties within the same species,indicating genetic background is a key determinant of fruit calcium uptake.The large sized pomes had the highest fruit calcium uptake rate,while the small size grape berries had the lowest;arillate fruits had the highest calcium uptake activity,while grape berries had the lowest.The phase pattern of fruit calcium uptake differed among species.‘Shatangju'tangerine and‘Shine Muscat'grape accumulated calcium chiefly in the early stage,while in most other fruit highest calcium uptake occurred during rapid fruit growth phase.In all fruit types,fruit calcium activity was highest during the early stage and decrease with fruit growth.A cross-species analysis showed that fruit calcium uptake rate was significantly correlated to fruit growth rate,indicating that demand for growth is the major factor determined fruit calcium uptake capacity.(2)Across species,calcium concentration was always significantly higher in the pedicel than in the fruit.The pedicel-fruit calcium concentration gradient is thus universal and was found to generally enlarge with fruit maturation.There was a significant positive correlation between calcium concentrations in the pedicel and in the fruit.Hence,the more calcium transported to the fruit,the more calcium stays in the pedicel,suggesting universal existence of“bottleneck effect”in calcium transport at pedicel-fruit joint.Calcium in the pedicel was shown to be distributed more in the phloem,where a large number of calcium-rich bodies were found.Structural calcium and oxalate calcium were the major forms of calcium in the pedicel.During fruit development,concentration and relative content of calcium in different forms fluctuated,suggesting inter-conversion among calcium forms.In arillate fruit,oxalate calcium was a major form of calcium in the pedicel.There was a significant positive correlation between oxalate calcium concentration and calcium concentration in fruit in litchi and longan,although in other species,no correlation was found.Our results indicated that formation of calcium oxalate did not cause the“bottleneck effect”.(3)Girdling made on bearing shoots suppressed fruit calcium uptake as well as fruit growth.Introducing La via bearing shoots also reduced fruit growth and calcium uptake,suggesting involvement of calcium channel in fruit calcium uptake.Feeding calcium analog Sr to fruit via peduncle showed that Sr was mostly distributed in the phloem in the pedicel similar to calcium distribution.The results proved that phloem is an important pathway for calcium transport to fruit.At the same time,they further prove the close interrelations between fruit growth and calcium uptake.(4)Fruit transpiration and xylem sap inflow was generally highest in the early stage and decreased with fruit development.This pattern generally agrees with results of dye-visualized xylem functionality.They displayed a significant positive correlation with fruit calcium uptake activity,suggesting fruit transpiration is favorable for long distance transport of calcium towards fruit.However,there were some special cases in individual species.‘Tengmu No.1'apple lose xylem functionality at early fruit stage,therefore its fruit calcium uptake might depend on phloem pathway.In contrast,the arillate fruit of litchi and longan had good xylem functionality through fruit development.Despite that,as shown in litchi,phloem pathway still contributed a major part of fruit calcium uptake.‘Zaozhong No.6'loquat showed poor xylem functionality at early and late fruit stages.Vessel size and density were not limiting indexes for fruit calcium uptake.(5)In arillate litchi and longan,fruit IAA level was significantly positively correlated to fruit calcium uptake.Exogenous auxin,2,4-D,promoted fruit calcium uptake as well as fruit growth in longan.In all species,a strong positive correlation between seed size and fruit calcium uptake was found.The results suggest an internal mechanisms regulates fruit calcium uptake capacity.(6)Calcium transporters(Ca²⁺channel,Ca²⁺-ATPase and Ca²⁺exchanger)provide mechanisms for positive uptake or movement of calcium in plants.According to the reported sequences of the three types of calcium transporter proteins,1 TPC1(Lc TPC1),1ACA(Lc ACA-2)and 2 CAX(Lc CAX1a-1and Lc CAX1a-2)genes were screened from litchi genome database.Gene expression of Lc TPC1 and Lc ACA-2 in the pedicel and pericarp increased with the growth and development,coinciding with the calcium accumulation pattern in the two tissues.VIGS significantly reduced the gene expression levels of Lc TPC1and Lc CAX1a-2 in various tissues as well as calcium concentration in the pedicel and total calcium content in the fruit.Silencing Lc TPC1 also significantly decreased fruit growth rate and fruit calcium uptake rate,while silencing Lc CAX1a-2 just slightly reduced fruit growth and calcium uptake rates.Interestingly,TRV-Lc CAX1a-1 treatment targeted to silence Lc CAX1a-1 failed only slightly reduced gene expression but significantly decreased fruit growth rate,calcium uptake rate and the calcium uptake activity of the fruit.The results suggest that both calcium channels and vacuolar Ca²⁺/H⁺exchange transporters are directly in fruit calcium uptake.