Investigations Of The Effects Of Zn Biofortification In Different Wheat Genotypes

Zinc(Zn)is one of essential elements for human,animal and plants.More than 30%of the world's population suffer from malnutrition caused by Zn deficiency,which is seriously harmful to human health.People take in Zn and other elements through food.Common wheat(Triticum aestivum L)is a staple food for nearly 40%of the world's population,however,most of wheat products have much lower levels of Zn than the recommended minimum intake threshold of 40mg kg-1 either due to the low abundance of available Zn in the soil in wheat growing regions or to the genotypic inefficiency in uptaking Zn from the soil.Thus,improving Zn nutrient in staple food by Zn biofortification,especially in wheat flour,has become one of major concerns for agronomists and nutritionists worldwide.Although several studies on Zn biofortification,either by root and foliar application,were reported and encouraging achievements have been made,these studies somewhat disagreed with each other in the effects of Zn biofortification on the quality traits as well as in the interactions,in a way of synergism or antagonism,between Zn and other elements(Ca,Mg,Cu,Fe,Mn,etc)in different plant tissues,and one of the reasons for the discrepancies from different studies in the interactions could be that only a few but different varieties were involved in these studies,and the heterogeneity of ecological environments,the soil type,fertilization strategy and farming management may also contribute to the complexity of Zn biofortification.In addition,few reports have been proposed about the comprehensive assessment of the potential effects of Zn biofortification on the distribution and accumulation of mineral elements in different tissues,and on processing quality(the gelatinization characteristics of the flour,etc.)and nutritional quality(protein content,wet gluten,etc.).In this study,we evaluated variations of Zn contents over two consecutive years,both in the control and soil application of Zn,in a panel of 104 wheat varieties from various sources,and established associations of Zn concentration in the seeds with genome-wide Single Nucleotide Polymophism(SNP)markers,which could be useful in improving Zn concentration by marker assisted selection(MAS).Forty of 104 varieties were then randomly selected for further investigation of the effects of Zn biofortification by foliar spray on the concentrations of six elements including Zn,Ca,Mg,Cu,Fe and Mn,wheat quality traits and physiological characteristics.Then a panel of ten varieties with distinct levels(high,medium and low)of Zn concentrations were biofortified with four gradient levels of Zn fertilizer at two developmental phases to understand the changes of the six elements across above-ground tissues/organs.The followings are major findings:1.One hundred and four tested varieties showed significant genotypic differences in Zn contents in wheat grains,ranging from 20.17 mg kg-1 to 55.44 mg kg-1 under natural conditions,and the mean value of Zn contents in wheat grains was lower than the recommended minimum intake dosage of 40mg kg-1 for human per day.Zn concentrations in wheat grains were significantly increased by Zn biofortification by soil fertilization,ranging from 25.49mg kg-1 to 63.83mg kg-1.2.Zn contents in wheat grains of 40 varieties were increased significantly after foliar spray and the amplitude of increase varied with genotypes,and Zn contents in wheat grains were higher than 50mg kg-1 in 70%of the varieties,and 37.5%of the varieties were higher than 60.5mg kg-1,and the amplitude of increase varied from genotypes and were not subjected to Zn contents in wheat grains of the controls.Foliar spray of Zn exerted either synergistic or antagonistic effects on the other five covalent minerals,generally decreasing the average contents of Fe and Ca in wheat grains and increasing the average contents of Mg and Mn,with no significant effects on the average content of Cu.However,there were genotypic differences in the responses of different wheat varieties to Zn biofortification.Fe and Ca are two beneficial elements to humans,and when compared with the controls,22 wheat varieties showed somewhat decreases and 18 showed increases in grain Fe content,and 26 wheat varieties exhibited somewhat decreases and 14 showed increases in grain Ca content after foliar spray of Zn treatment.Four varieties including Yangmai2,Yangmai4,Yangmai6 and Bobwhite showed a synergistic increase of Zn,Fe,Ca,Mg,Cu and Mn concentrations in grains after Zn treatment.The concentrations of four of the five elements(Ca,Fe,Mg,Cu,Mn)showed somewhat increases after Zn treatment in the varieties of Wheaton,HFZ,Yangmai1,Yangmai5,Yangmai10,black-colored wheat,6209 and Lumai21,and these varieties proved to have good potential for Zn biofortification.3.Association analysis of Zn concentration in grains(the control,Zn treatment,and the difference between Zn treatment and the control)with genome-wide single nucleotide polymorphism(SNP)markers were performed,and we found that the SNP markers on chromosomes 4A,5A,6A,2B,3B,7B and 4D were repeatedly associated with Zn concentration in grains,and these markers explained 7.3%-15.7%of phenotypic variations in Zn content,suggesting that there were quantitative trait loci(QTL)responsible for Zn content on these chromosomes.The QTL on 3B and 7B might be novel QTL that have not been reported before,and the favorable alleles at QTL on 7B had a larger effect on increasing Zn concentration in grains.4.There were significant genotypic differences in mineral contents of flour and bran between different wheat varieties.Bran had a 3-19 folds of Zn,Ca,Fe,Mg,Cu and Mn than those that flour had in all the varieties.Mg content in bran was almost 19 folds of that in flour and Fe content three folds of that in flour.The sequential trend of elemental contents in flour was:Ca>Mg>Fe>Zn>Mn>Cu,and that in bran was Mg>Ca>Fe>Mn>Zn>Cu.The responses of different elements in bran and flour to Zn varied from varieties after Zn treatment.The contents of Zn in flour of all varieties showed somewhat increases while the responses of Fe,Cu,Mn,Ca and Mg contents depended on the genotypes.The ratios of Ca,Fe,Mg,Cu and Mn contents in flour to those in bran were 0.61,0.68,0.16,0.43 and 0.14,respectively.Bobwhite and Luozhenl had relatively higher contents of Zn,Ca,Fe and Cu in flours;Zimail had higher contents of Mg,Cu and Mn in their respective flours.South Korean variety Chokwang had higher contents of Mn and Fe in the flour.Higher contents of Mg and Fe were observed in the flour of Jingzhou D402.All of the three colored wheat varieties,Zimai1,Luozhen1 and black-colored wheat had higher elemental contents than the average value of all the varieties.5.Foliar spray of Zn resulted in significant or highly significant changes on viscosity(RVA)characteristics of wheat flour.Significant or highly significant differences were observed between the control and the samples in the five RVA characteristics including peak viscosity,breakdown,trough viscosity,final viscosity and setback,indicating Zn treatment may alter the food processing characters of wheat.D402,Yangmai6 and Yangmai20 exhibited somewhat increases in peak viscosity and breakdown of after Zn treatment.There were significant differences in protein and starch contents among varieties,and the protein content of wheat grains showed a significant increases after Zn treatment.Different varieties responded differentially to Zn biofortification in protein content,and six varieties including Wheaton,HFZ,Yangmai4,Yangmai5,Yangmai6 and Chokwang showed an significant increase in protein content after Zn biofortification.Foliar spray of Zn did not give rise to significant changes in total starch content.The response of wet gluten content to Zn treatment varies among varieties.Bobwhite and Chinese landrace HFZ ranked top two in wet gluten content both in the control and Zn treated samples,and the latter had the largest increase in wet gluten content after Zn treatment.6.The contents of all the six elements including Zn,Ca,Fe,Cu,Mg and Mn in stem,leaf,grain and glume&rachis,respectively,showed highly significant genotypic differences.Significant differences in Zn concentration were observed among four gradients of Zn fertilizer dosages as well as between the two developmental phase of spray,and the effects of interactions of variety-phase-Zn dosage on Zn concentrations in different organs(or tissues)were also significant.Zn treatments changed the distributions of the elements in different organs.Zn contents in all the organs were significantly increased after Zn treatments both at the flowering stage and 10 days after flowering(DAF),when compared with their respective Zn concentrations in the controls.However,the increase ranges in Zn concentration in the organs did not show linear increases with the gradient dosages of Zn fertilizer.In general,despite of the organs,sprays at 10 DAF across the three Zn dosages resulted in higher Zn concentrations than those at the flowering stage.The sequence of Zn contents was showed as leaf>glume&rachis>grain>stem both in the controls and in the samples with low dosage of Zn fertilizer while the sequence of Zn contents in organs was showed as leaf>glume&rachis>stem>grain with higher dosages of Zn treatment.Ratios in Zn contents of leaf/kernel,glume&rachis/kernel and stem/kernel increased with the increase in dosages of Zn fertilizer,indicating more Zn were contained in the organs of leaf,glume&rachis and stem,rather than in the grains.Among the ten wheat varieties,Bobwhite,HFZ,Chokwang and Hexuan198 had lower ratios of leaf/kernel,glume&rachis/kernel and stem/kernel in Zn concentration,suggesting these four varieties had better potential of Zn biofortification.Spray at the flowering stage had more effects on Ca contents in the glume&rachis and stem than spray at 10 DAF.Zn treatment decreased Ca content in leaves while it increased Ca content in grains.sprays both at the flowering stage and 10 DAF significantly decreased Mg contents in grains and stems,and spray at 10 DAF gave rise to more reduction of Mg content in grains.More Mg was kept in leaf and grains with treatment with 0.3%at 10 DAF.Cu contents in glume&rachis decreased after spray of Zn fertilizer both at flowering stage and 10 DAF.Cu content in stem was decreased significantly after spray at 10 DAF while it was increased at the flowering stage,but did not show significant differences in the concentration.Cu content in leaf was increased after spray at flowering stage while it was significantly decreased after spray at 10 DAF,and Zn treatments both at the two time-points increased Cu content in grains.Fe contents in glume&rachis,leaf,and grain were decreased after Zn treatments at both stages.Mn contents in all of above-ground organs were decreased with different Zn treatments at both stages,and the degree of reduction varied from combinations of spray time and Zn dosage.The facts above indicated that exogenous Zn treatment alters the distribution patterns of other elements in organs,and may help Ca,Mg and Cu move from stem and leaf to grain while enhance the transportation of Fe and Mn from above-ground organs to root or to soil medium.7.Wheat varieties showed highly significant genotypic differences in leaf SPAD values.Application of Zn did not show significant effect on SPAD value of leaf.In addition,Zn contents in grains did not show significant correlations with leaf SPAD value.In general,SOD and CAT in wheat varieties showed a slight decrease while POD exhibited a slight increase after Zn treatment,but significant changes in enzymatic activities of SOD,POD and CAT were not observed between the control and Zn treatment.In summary,Zn biofortification had multiple impacts on grain nutritional quality such as mineral nutrition,protein content etc.as well as wheat processing quality such as pasting viscosity and wet gluten etc.Therefore many factors should be taken into consideration and comprehensive evaluations should be done.In addition,since almost all the traits are genotype-dependent,wide germplasm resources including wheat landraces,elite commercial cultivars and foreign accessions should be collected and used to improve Zn nutrition and other beneficial characters,when combined with markers or favorable alleles associated with Zinc concentration,ideal genotypes could be obtained to meet the increasing demand of enhanced mineral nutrition(Zn,Fe,etc),and other beneficial characteristics such as protein content and processing quality for specific purposes.