| Wheat(Triticum aestivum L.)is one of the main staple crops in the world.It is the second food crop after rice in China,and plays a very important role in the national food production.The future development of the Chinese wheat production will be implemented by the path of reducing the cost and improving the grain yield,because of the global warming,population growth and the decrease of agricultural resources.Starch,the main component of wheat grain,is closely related to the grain yield and the quality of flour.Drought is one of the major factors,causing the loss of wheat grain yield and the deterioration of flour quality.Furthermore,drought stress during grain filling could reduce starch accumulation,and influence the structure and components of starch granules.However,it is unclear for the mechanisms of physicochemical characteristic changes within surface of starch granules under drought stress in wheat.In this study,two wheat varieties were used to reveal biological mechanisms of drought on starch granules through methods of cell biology,plant physiology,molecular biology and histochemical analyses.The main results are as follows:1.Scanning electron microscopy(SEM)revealed the formation of pits and pores on the surfaces of starch granules in response to drought stress.Drought induced a significant reduction of average sizes of starch granules in both wheat varieties,by 11.74% in Xindong 20,and 14.55% in Xindong 23,respectively,compared with their respective controls.Under drought stress,the concentrations of reducing sugars after digestion with ?-amylase and amyloglucosidase were significantly increased,by 38.60% and 23.56%,respectively,in Xindong 20,and increased by 12.84% and 18.06%,respectively,in Xindong 23,compared with their respective irrigated controls.The increasement of numbers and extents of pores and channels within granules after drought treatment may be the potential sites to facilitate the transfer or improved access to granules for enzymes.Confocal laser scanning microscopy(CLSM)revealed that starch granules from the two wheat varieties grown under drought conditions had substantially increased fluorescence after treatment with proteolytic enzymes and staining with methanolic merbromin and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde dyes.2.Under drought stress,the typical A-type crystals of wheat granules were not changed;however,the degrees of crystallinity were decreased by 15.61% in Xindong 20 and 3.73% in Xindong 23,respectively,compared with their respective irrigated controls.The peak signal intensity was decreased in amylopectin starch sugar.Meanwhile,the number of chromatographic peaks was losted under the drought post antesis.Drought induced a significant reduction in grain weights,total starch contents,amylose contents and amylopectin contents in both wheat varieties,decreasd by 19.61%,28.71%,28.17% and 28.94%,respectively,in Xindong 20,and decreasd by 17.49%,4.57%,4.47% and 4.62%,respectively,in Xindong 23,compared with their respective controls.Analysis of pasting properties showed significant increases of peak viscosity,trough viscosity,break down,and setback following drought stresses.Furthermore,drought induced a significant reduction in the water binding capacity and increased damage to starch only in Xindong 23.3.In response to drought stress post anthesis,the gene expression profilings of wheat grains at 21 days post anthesis(DPA)were different based on the transcriptomic sequencing.There were 1368 and 194 differentially expressed genes in Xindong 20 and Xindong 23,respectively.Among these 1368 genes in Xindong 20,there were 998 up-regulated genes and 370 down-regulated genes.Among the 194 genes in Xindong 23,there were 67 up-regulated genes and 127 down-regulated genes.To classify differentially expressed genes of the two wheat genotypes,they were functionally classified to metabolic process,cell metabolism,biological regulation,and response to stimulus.The up-regualted genes were prominent among the starch breakdown and sucrose metabolic process.The down-regualted genes were prominent among the starch biosynthesis and sucrose metabolic process.4.Under the drought stress,the relative expression of starch synthase genes including agp I,agp II,ss I,ss II,ss III,sbe I and sbe IIb,and the enzyme activities of AGPase,SS and SBE tended to decrease in Xindong 20 and Xindong 23 compared with their respective irrigated controls.In response to drought,?-amylase and ?-amylase activities were increased in both wheat varieties,compared with their respective irrigated controls.However,the relative expression patterns of starch degradation enzyme-encoding genes depend on the genotype.Drought induced an up-expression of amy I,amy II,amy III,amy IV,bam I and bam V genes in Xindong 23,whereas under drought stress,the relative expression of amy I,amy II,amy III,amy IV and bam V genes was reduced in Xindong 20,compared with their respective irrigated controls.These results were in agreement with the increased pits and channels on the surfaces of starch granules in Xindong 23.In-situ hybridization analyses revealed the spatio-temporal profilings of starch degradation genes;amy IV,bam I and bam V genes were mainly expressed in pericrap,aleurone layer and nucellar cells at 7 DPA.With the proceeding of filling(7 DPA-28 DPA),expression sites of the genes expanded to the central endosperm.At the late stages of filling the expression sites of the genes were distributed throughout the endosperm.5.Taken together the above-mentioned results of starch granule development,microstructure,starch quality characteristics,and the spatio-temporal profiling of starch biosynthase and degradation enzymes,we proposed a hypothesis that the increased pitting of the granule surfaces observed when wheat is exposed to drought after anthesis is a consequence of an enhanced action of ?/?-amylase enzyme activities in the endosperm.The drought stress may disturbe the balance between starch biosynthase and degradation enzymes in the grain filling stages,thus reducing the activities of starch synthase.Overall,these results indicate that drought has important impacts on starch properties and that the genetic background of a given wheat variety plays a decisive role in the differential responses of starch accumulation in wheat in response to drought.This study may serve as a foundation for further basic investigations into the mechanism(s)underlying drought tolerance and susceptibility and should prove beneficial in the breeding of drought tolerant wheat varieties. |
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