| Two soft wheat cultivars ‘Xuke 316' and ‘Huapei 8'(SKCS and hardness index were 30 and 41%,respectively)and one hard wheat cultivar ‘Zhengmai 366'(SKCS and HI respectively 80 and 75.9%)was used to study the effects of starch surface polar lipids on the binding of PINs to starch surface and the effect on grain hardness by in vitro protein binding method and flour recombinant method.At the same time,change of starch surface lipid content,PINS protein expression and the grain hardness of wheat grain under different drying methods(drying and freeze-drying)during grain filling were investigated.Additionally,endosperm microstructure,starch granule,amyloplats and amyloplast membrane were observed by electron microscopy.The main results are as follows:1.The role of starch surface polar lipids in the binding of PINs protein to starch surface and the relationship with the endosperm structure and hardness1.1 The results of SDS-PAGE showed that 15 k D band was shown in soft wheat and hard wheat starch,but no corresponding band was observed in starch treated with proteinase K,defatted-starch,and defatted-starch combining with PINS protein.But,there was 15 k D band in the treatment,which defatted-starch combining with PINS protein after adding starch surface polar lipids.These results proved that the starch surface polar lipids play an important role in the binding of the PIN protein and starch granules.1.2 The reconstituted flour was pressed into a dry flour tablet with a tablet press.At the same time,the reconstituted flour was made to dough and then the dough was pressed into a certain thickness of dough sheet.The hardness of flour table and dough sheet was measured by TPA.Hardness of flour table and dough sheet shared the same change trend among different treatment.Hardness of Gluten+defatted-starch is higher than that of Gluten+Prime starch.Group Gluten+ Defatted-starch + lipids +PINs had the lowest hardness,which indicated that polar lipids and PINs protein block the binding ability of starch granules and gluten.It proved that polar lipids and PINs protein content decreased after de-fatting and starch granules combined with gluten increased the hardness.However,polar lipids and PINs protein blocked the binding of starch granules and gluten after the addition of polar lipids.1.3 The internal microstructure of flour tablets and dough sheets was observed by scanning electron microscopy.There are no obvious differences between Gluten+defatted-starch and Gluten+Defatted-starch+PINs whether at the flour tablets or dough sheets.After lipid addition,there were small aggregates on the starch granules surface on flour tablets;and starch granule were embedded in gluten on dough sheets.These results indicated that lipids play a function in binding role between the starch granules and the PINs proteins.2.The role and mechanism of starch surface polar lipids in the formation of grain hardness2.1 The results showed that hard wheat and soft wheat got corresponding hardness value(oven drying)for distinguish soft and hard wheat at the early grain filling stage.Two varieties all showed the higher grain hardness under oven drying than freeze-dried.The biggest difference between two dry methods for ‘Zhengmai366' was observed at 14 DAS,and the hardness of the oven-dried grains was 34 higher than that of the freeze-drying ones.2.2 Respectively.DGDG,MGDG and total lipid content of ‘Xuke316' is slightly higher than that of ‘Zhengmai366'.If the comparison was made between the two kinds of drying methods,there is no significant difference for polar lipid content of whole meal.Under two kinds of drying method,the starch surface polar lipid content of ‘Xuke 316' were significantly higher than that of the corresponding‘Zhengmai366'.‘Zhengmai366' got the higher polar lipids contents under freeze-dried drying method than that of under oven drying method(except for DGDG at 28 DAS);while ‘Xuke316' got a higher polar lipids contents under oven drying method only at certain grain filling stage.These results indicated that the starch surface polar lipid content was negatively correlated with grain hardness.2.3 Total PINs protein of whole meal of soft wheat ‘Xuke316' was slightly higher than that of‘Zhengmai366'.The expression of PINs had no significant difference between two kinds of drying methods.The starch surface PINs protein contents of ‘Xuke316' were significantly higher than that of‘Zhengmai366' during grain filling stage.The expression of starch surface PINs protein of two wheat varieties all showed a higher value under oven drying method than that of under freeze-drying.These results showed that grain hardness was negatively correlated with the starch surface PINs protein content.However,the decrease of hardness under freeze-drying may be not related to the expression of Pins protein on starch surface.2.4 It was observed that wheat starch granules were deeply embedded in the protein matrix,and there are close combination between starch and protein in the hard wheat variety ‘Zhengmai366' by scanning electron microscope.However,the combination between starch and protein of soft wheat is not close and the endosperm structure texture is loose.If the comparison was made between two different drying methods,the combination was relatively loose under the freeze-drying method than that of under oven drying method.The inter microstructure of TEM found that fresh seed cell structural is integrity,cell wall architecture outline is clear and complete,the amyloplast and proteoplast is clear,and amyloplast membranes is complete.Amyloplast membranes are separated with starch granules with the grain dehydration.The separation of amyloplast membranes with starch after oven drying degree 1/3 were larger than that of under freeze-dried 1/3 method.Under oven drying 30 percent,large amyloplast membranes were separated with starch granules,and small amyloplast membrane were separated with starch granules partly,and membrane is not complete,partial and visible.But,the grain after treatment with 1/3 freeze-drying have more protruding vesicles,no obvious amyloplast membrane and starch separation,and amyloplast membrane is locally visible.The similar trend was also showed in the hard wheat variety under different drying methods;hard wheat had more separation between amyloplast membrane and starch granules,and the treatment of oven drying 1/3 got a more apparent separation.The results showed that the separation of amyloplast membrane with starch granules may reduce membrane lipid adhering on the surface of starch,then affect the grain hardness.2.5 Front-face fluorescence analysis showed that the fluorescence intensity of soft wheat flour and starch were higher than that of hard wheat,but the difference between starches is more apparent.The fluorescence intensity of soft wheat starch at 14 and 35 DAS under freeze-drying method is higher than that of under oven drying method;while hard wheat got higher value under freeze-drying than that of under oven drying during grain filling stage.The similar tendency of fluorescence intensity and polar lipids contents during grain filling and under different drying method indicated that polar lipids contents,and its binding character with PINs protein would influence grain hardness. |
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