Influence Of Powdery Mildew On Quality And Its Molecular Mechanism In Wheat (Triticum Aestivum L.)

Powdery mildew is one of the most severe fungal diseases responsible for yield reductions in wheat. No systematic report has been found on the influence of powdery mildew on qualities and its molecular mechanism in wheat. Because wheat grains would develop various mechanisms for stress adaptation, our analysis on the accumulation and qualities of storage materials (mainly starch, protein and soluble sugar) would benefit the study on the mechanism of powdery mildew on the changes of grain qualities. Experiments were conducted in the Science Education Experimental Park of Henan Agricultural University from 2009 to 2011 using a split plot design with four replications. The main treatment was powdery mildew severity, which was respectively marked as T1, T2 and T3. T1 was the control, T2 and T3 was the severe and the moderate powdery mildew separately. The secondary treatment was the series of wheat varieties. A series of powdery-mildew-infectious varieties were applied to grow in 2009-2010. There were 5 susceptible varieties, including Xi’nong 979 and Zhengmai 9023 of strong gluten; Zhoumai 18 and Zhongyuan 98-68 of moderate gluten; and Zhengmai 004 of weak gluten. Finally, Guomai 301 was selected as the control powdery mildew-resistant material. In 2010-2011, Xi’nong 979, Zhoumai 18, and Guomai 301 were used for continuity; in addition, Zhoumai 22 of high resistance was included. At anthesis, the ears were sampled and shelled for grains every five days. Some samples were used for the analyses of protein and starch contents as well as the concentrations of their compositions. Other samples was also used for metabolic protein analysis by 2-DE and MS/MS. Mature samples were also used for technology analysis such as viscosity, swelling power and falling number as well as sedimentation value, extensograph and farinograph parameters. The study aims to gain an insight into the influence of the disease on grain quality and the molecular mechanisms, in the hope of providing theoretical basis for improving grain qualities in practical production.The main results obtained from the above efforts were as follows:1. Effects of wheat powdery mildew on grain protein and it’s compositions such as albumin, globulin, gliadin and glutenin were analyzed. (1) Powdery mildew’s influence on total quantity of metabolic protein of disease-infected varieties didn’t achieve significant level. However the disease caused remarkable increase of the total quantity of storage protein and further led to remarkable rise of total protein concentration. This revealed that the infection of powdery mildew could change the protein composition of wheat grains. (2) The turning point of glutens with significant increase was found after 20 DAA. At 20 DAA, when the disease reached the greatest severity in the field, concentrations of gliadin and glutenin increased significantly with the increase of disease indices (DI). Results showed that wheat powdery mildew had great influence on the accumulation of gliadin and glutenin in grains. (3) The difference of grain albumin concentration among different treatments wasn’t significant. This indicated that the totality of various metaboloc enzymes didn’t have significant differences. Significant increase of globulin accumulation was found at 20-25 DAA stage, and there were different increasing amplitude among different cultivars with different DI changes and different developing stages. With the disappearance of powdery mildew symptoms at 30 DAA to maturity, globulin concentration restored to a constant level. The study also showed that the differences of grain protein concentration and its compositions concentrations among disease-resistant varieties were not significant.2. Proteomics analysis on metabolic protein expression was performed by using Xi’nong 979 grains at the two growth stages of 10 DAA and 25 DAA under the stress of powdery mildew. This analysis was carried out to study metabolic protein’s reaction to the infection of powdery mildew. Results showed that the metabolic protein in grains changed significantly with the infection of infectious varieties.38 proteins were identified by mass spectrum analysis. Among them, enzymes or molecular chaperones taking part in protein synthesis (such as Aspartate carbamoyltransferase OS, single-stranded nucleic acid binding protein, Pantothenate synthetase OS, GTPase ObgE, cyclophilin A-2, peptidylprolyl isomerase) all increased. All of them took part in the courses of mRNA transcription and protein translation as well as courses of protein folding and post-translational modification. The rise of enzymes or molecular chaperones at the two growth stages of 10 DAA and 25 DAA agreed with the rise of total protein content during the same stage. However, proteins involved in life processes like glycolysis, starch biosynthesis, and the tricarboxylic acid cycle (such as β-amylase, plasmid α-1,4-glucosan phosphorylase, Sucrose synthase type 2, glyceraldehyde-3-phosphate dehydrogenase), all showed a decreasing or even disappearing trend. This implied that the synthesis of starch in mildew-infected wheat had been inhibited. Disease resistance proteins like NBS-LRR type RGA and GTPase, as well as defense proteins like Pm3b-like 2Q9, Tri a Bd 27K, peroxiredoxin and cold regulated protein were also indentified in this study. This revealed that interactions resulting from defense/stress proteins among proteins took place. Enzyme quantity of defense/stress increased at 25 DAA compared with at 10 DAA. This showed that grain at its developing stage before and after 25 DAA responded much intensively to the stress environment, which agreed with the highest disease indices of powdery mildew at this stage in the field. This study has discovered that some non-gluten storage proteins, such as globulin 3, globulin 3B, globulin 3C and gliadin/avenin-like protein increased, which agreed with the increase of total globulin quantity at this stage, indicating that powdery mildew infection promoted the synthesis of some specific globulins in wheat grains.3. Effects of wheat powdery mildew on grain starch and it’s compositions as well as soluble sugar were analyzed. (1) The two-year results showed that concentration differences of amylose, amylopectin and total starch as well as soluble sugar were quite significant among disease-infected varieties. The amylose concentration increased, while amylopectin and total starch concentration decreased with the increase of powdery mildew severity. Therefore amylose/amylopectin increased significantly. It indicated that Bgt infection had an influence on grain starch compositions. Meanwhile, soluble sugar concentration decreased significantly with the increase of disease indices. (2) Wheat powdery mildew had a significant effect on starch accumulation. The turning point of amylose concentration with significant increase was found at 20 DAA when the disease reached the greatest severity in the field, the amylopectin and total starch accumulation decreased significantly from 10 DAA to mature. In addition, the changing tendency of total starch concentration was found to be similar to that of amylopectin. Therefore amylose /amylopectin increased significantly. (3) Soluble sugar concentration was sensitive to powdery mildew affection. At early dynamic stages, soluble sugar increased before 25 DAA. However, the following tendencies varied among different cultivars, that is, soluble sugar concentration of susceptible cultivars decreased after 25 DAA until mature, and that of moderately susceptible/resistant cultivars kept on increasing. But the soluble sugar accumulation of disease-resistant varieties didn’t achieve significant level. (4) Analysis of metabolic protein expression demonstrated the starch-synthesis-related enzymes decreased during Bgt infection, glyceraldehyde-3-phosphate dehydrogenase involved in life processes like glycolysis and the tricarboxylic acid cycle, and P-amylase, plasmid α-1,4-glucosan phosphorylase, Sucrose synthase type 2 involved in starch biosynthesis, all decreased. This implied that the synthesis of starch in powdery-mildew-infected wheat had been inhibited.4. Effects of wheat powdery mildew on processing qualities, such as SDS sedimentation value, farinograph and extensograph parameters as well as viscosity, etc were studied. (1) Results showed that wheat powdery mildew had significant influences on starch quality and that different cultivars had similar changing trends. Under Bgt infection, grain weight decreased, which indicated the decrease of grain plumpness. Meanwhile swelling power decreased, indicating the decrease of water holding capacity of flour. With the increase of disease indices, falling number and viscosity increased, indicating that a-amylase activity decreased. (2) With the increase of disease indices, some parameters increased and these parameters included SDS sedimentation value, the dough development time, stability time, farinose evaluation value and extensibility. However, extension resistance and extension ratio declined. The changes among strong-gluten-type wheat cultivars reached a significant level.5. The effects of powdery mildew on grain yield and quality yield were analyzed. The study showed that grain yield, grain starch yield and protein yield decreased significantly with the increase of powdery mildew disease indexes. Among them, starch yield suffered the greatest decline, while protein yield suffered the smallest decrease. This indicated that wheat powdery mildew could not only decrease economic yield, but also modify grain quality through changing the relative content of storage materials. Further analysis indicated that yield reduction range varied according to different cultivars and different disease severities. It was also found that the reduction amplitude of grain weight increased with the rise of disease severities.To sum up, following conclusions could be drawn in this study:(1) Powdery mildew has great influence upon the nutritional quality in grains. The infection of this disease would increase protein (especially storage protein) concentration, but decrease starch (especially amylopectin) concentration. (2) Powdery mildew could affect the processing qualities of grains.The changing tendency of starch qualities was found to be similar to that of starch concentration and the changing tendency of protein also consists with that of protein accumulation. (3) The quality changes caused by the influence of powdery mildew may result from the changes of metabolic enzymes expression. The upregulation of protein-synthesis-related enzymes and molecular chaperones consists with the rise of storage protein concentration and with the changing tendency of protein quality. Meanwhile the downregulation of starch-synthesis-related enzymes also accords with the fall of starch concentration and with the changing tendency of starch quality.