Localization Of Ca²⁺ And Related Enzymes In Phloem In The Developing Caryopsis Of Triticum Aestivum L.

Sieve elements(SEs) in the ventral vascular bundle of wheat(Triticum aestivum L.) caryopsis are considered to be the major conducting channels for photo-assimilates transportation.The formation of conducting channels and the conducting rate of photo-assimilates transportation are related to the accumulation of photo-assimilates in wheat caryopsis,as well as the wheat yield and quality.Previous study revealed that SEs in the developing caryopsis of wheat underwent a unique type of programmed cell death (PCD).This study was carried out on wheat(Triticum aestivum L.cv.Huamai 8) caryopsis.The ultrastructural aspects of phloem cells in wheat caryopsis were observed by transmission electron microscopy.Using specific fluorescence staining and potassium pyroantimonate precipitation method,Ca²⁺ were localized at histological and sub-cellular levels in phloem in the developing wheat caryopsis.Transmission electron microscopy and lead nitrate were used to locate Ca²⁺-ATPase and ATPase in phloem.The different photo-assimilates in grains were determined by Anthrone's method during grain filling of wheat.We aimed to investigate the dynamic changes and the roles of Ca²⁺,Ca²⁺-ATPase and ATPase in phloem during SEs differentiation,to analyze the relationship between ATPase and the accumulation of photo-assimilates in grain during the filling stage.The main results were as follows:1.Transmission electron microscopy studies showed that the cell walls of SEs thickened at the beginning of differentiation,and then became thinner and smoother.2.Fluorescence staining showed that the fluorescence due to Ca²⁺ appeared in cell walls of SEs from 6 to 10 d after flowering.The fluorescence due to Ca²⁺ in cell walls of SEs was most notable at 9 d after flowering and disappeared at 14 d after flowering.3.Sub-celluar localization of Ca²⁺ showed that Ca²⁺ was localized on plasmalemma and in nuclei from 1 to 2 d after flowering.At 4 d after flowering,Ca²⁺ was localized in cytoplasm and mitochondria of SEs.From 5 to 6 datter flowering,Ca²⁺ was transported to the cell walls of SEs and no Ca²⁺ precipitates were observed in mitochondria.From 10 to 18 d after flowering,Ca²⁺ was transported into the cytoplasm again from the cell walls.In intermediary cells(ICs),Ca²⁺ precipitates were observed from 1 to 18 d after flowering,and Ca²⁺ mainly distributed on intine and tonoplast.4.Localization of Ca²⁺-ATPase showed that there was lowest activity of Ca²⁺-ATPase in SEs at 3 d after flowering.High levels of Ca²⁺-ATPase activity were observed in SEs from 4 to 14 d after flowering,and the enzyme was mainly localized in cell walls,cytoplasm,plasmodesmata and mitochondria.5.Localization of ATPase showed that there was lower activity of ATPase in SEs from 2 to 10 d after flowering,the enzyme was mainly localized on plasmalemma and plasmodesmata;from 14 to 18 d after flowering,there were higher levels of ATPase activity on the plasmalemma of SEs and lower ATPase activity in ICs;at 30 d after flowering,there was lower activity of ATPase on the plasmalemma of SEs and higher apparent ATPase activity on the plasmalemma of ICs.6.Through the measurements of photo-assimilate contents,14 d after flowering was considered to be the turning point during the filling stage.On one side, photo-assimilates were mainly accumulated in the form of soluble sugar before the turning point and in the form of starch thereafter.On the other side,the total sugar per grain showed a "S"-type growth.The accumulation rate of total sugar was very slow before the turning point(the accumulation rate slightly increased during 10-14 d after flowering) and the accumulation rate increased rapidly thereafter.The accumulation rate of total sugar decreased during the final stage of grain filling.These results showed the dynamic changes of Ca²⁺ and Ca²⁺-ATPase during SEs differentiation.Ca²⁺ might play important roles in SEs during PCD.In addition,Ca²⁺ and Ca²⁺-ATPase might be also related to cell wall thickening and functions of SEs.ATPase activity was related to the accumulation rate of photo-assimilates.