Study On Wheat Caryopsis Development And The Response To Nitrogen And Drought Stress

Wheat is one of the important cereal crops worldwide. According to the characteristics of production, transportation and accumulation of nutrient during caryopsis development, wheat caryopsis can be divided into nutrient transport tissues, nutrient storage tissues and pericarp tissue. The coordinate development of these three types of tissues jointly determines the development status of wheat caryopsis. Nitrogen is the most active nutrient element that affects wheat caryopsis development, and plays an important role in the formation of wheat quality traits. Drought is one of the most important abiotic stress factors, which seriously affect the growth and development of wheat. In this study, we investigated the development rules and the interrelations of various tissues of wheat caryopsis and the response to nitrogen and drought stress by using various methods, including various types of microscope observation, physiological and biochemical determination, physicochemical properties determination, quality analysis, transcriptome sequencing and proteomics analysis etc. The main results are as follows:1. Structural development of wheat nutrient transfer tissues and their relationships with nutrient storage tissue development(1) Nutrient transfer tissues in wheat caryopsis include maternal nutrient transfer tissues that were composed of vascular bundle, chalaza and nucellar projection transfer cells, and filial transfer tissues that consisted of aleurone transfer cells, endosperm transfer cells, endosperm conducting cells, and aleurone layer. (2) Mature wheat nutrient storage tissue belonged to filial tissue, which included embryo and center endosperm. (3) The nutrient unloaded to endosperm cavity was transported to filial tissues via three pathways. The first pathway was from endosperm cavity to embryo via aleurone transfer cells, endosperm transfer cells and endosperm conducting cells/tissues sorrouding embryo, the second pathway was from endosperm cavity to endosperm periphery via apoplastic space, and then from endosperm periphery to center endosperm and embryo via aleurone layer, and the third pathway was from endosperm cavity to center endosperm via aleurone transfer cells and endosperm transfer cells. (4) The development of these nutrient transport tissues revealed the pattern of temporal and spatial and was closely coordinated with process of embryo and endosperm development. (5) Compared with waxy wheat, non-waxy wheat presented more well- developed nucellar projection transfer cells, aleurone layer transfer cells and endosperm transfer cells, resulting in a higher grain weight.2. Wheat pericarp development and the accumulation and degradation characteristics of pericarp starch(1) Wheat pericarp functioned in production and temporary storage of nutrients. Based on the structures and physiological characteristics, the developmental process of pericarp was divided into four stages, i.e. growth, formation, extinction and maturation stages. There were significant differences in the development process and starch accumulation in the different parts of different wheat cultivars. (2) Compared with endosperm starch granules, pericarp starch granules mainly presented as multiple starch granules with irregular shape and small size and were stored in the amyloplast or chloroplast in the pericarp. The contents of mineral elements in pericarp, suck as calcium, zinc, iron and potassium, were higher than those in the inner endosperm. Expression level changes of AGPase, GBSS Ⅱ, and AMY genes were closely related to starch synthesis and degradation. Amylose contents in pericarp starch were lower and relative degrees of crystallinity were higher at later stages of development than at earlier stages. Short-range ordered structures in the external regions of pericarp starch granules showed no differences in the developing pericarp. (3) Compared with rice and maize, there were significant differences in the accumulation characteristic of starch granule in wheat pericarp. Small amyloplasts were accumulated in dorsal and abdominal regions of wheat epicarp while they were accumulated only in dorsal regions of epicarp of rice and maize. Starch granules were accumulated in chloroplast in wheat and rice pericarp while they were not found in maize pericarp developed in the darkness. Wheat pericarp starch granules were single or compound starch granules with spherical and irregular in shape while rice and maize pericarp starch granules were both compound starch granules with irregular and polyhedral in shape.3. Effect of nitrogen treatment on protein body development and spatial distribution of starch granule in wheat endosperm(1) Nitrogen treatment altered the accumulation pattern of protein bodies. The endosperm in control group contained many smaller protein bodies that were scattered in endosperm cells in an unordered pattern, whereas the endosperm with nitrogen treatment contained many larger protein bodies or protein aggregations that were concentrated in a certain region of endosperm cells. (2) Nitrogen treatment significantly increased the amount and relative area of protein body in wheat endosperm. However, the cultivars differed with the degree of response to nitrogen being cv. Xumai 30 (hard wheat cultivar)> cv. Yangmai 13 (soft wheat cultivar). These differences also varied in the position in endosperm in the order dorsal region> abdominal region. (3) There were remarkably differences in the distribution patterns of starch granules in different endosperm regions in respond to nitrogen. The distribution characteristic of starch granules in sub-aleurone of abdominal endosperm (SAE) was similar to that in sub-aleurone of dorsal endosperm (SDE) and the distribution characteristics of starch granules in center of abdominal endosperm (CAE) and center of dorsal endosperm (CDE) were similar, but the distribution of starch granules in endosperm transfer cell (ETC) was different from those in above four endosperm regions. The density of starch granules in endosperm regions was following the order SAE> CAE>SDE> CDE> ETC. For abdominal and dorsal endosperm, nitrogen treatment increased the density of A-and B-type starch granules in sub-aleurone region and increased the density of B-type starch granules in center region but decreased the density of A-type starch granules in center region and the numbers of A-type and B-type starch granules in ETC.The results suggested that nitrogen treatment at the booting stage increased the accumulation amount of protein body and the density of small starch granules in different endosperm region and decreased the density of large starch granules, but that the results varied in different regions of the wheat endosperm.4. Effect of nitrogen treatment on synthesis and composition of endosperm storage protein and processing quality of wheat(1) A total of 3,483 differentially expressed transcripts (DETs) and 169 differentially expressed proteins (DEPs) were found at 7 days after anthesis (DAA), and 3,084 DETs and 154 DEPs was detected at 18 DAA after nitrogen treatment. Some DETs were found to be involved in protein biosynthesis pathways. (2) The DEPs related to storage protein biosynthesis contained gliadin and glutenin subunits and most of them were up-regulated after nitrogen treatment at 7 and 18 DAA, respectively. Quantitative real-time PCR (qRT-PCR) analysis indicated that some of these gliadin and glutenin subunit encoding transcripts were differentially expressed and most of transcript-protein pairs showed similar expression tendency; (3) Correlation analysis showed that nitrogen treatment/control at 7 and 18 DAA respectively matched 1162 and 1063 transcript-protein pairs and the transcription of the transcript and protein expression level showed very low correlation. (4) Nitrogen treatment affected wheat protein composition and quality properties. Nitrogen treatment not only enhanced the gliadin, glutenin and storage protein contents in wheat caryopsis during different developmental stages but also promoted the processing qualities including gluten content, water absorption, stability time and elasticity properties.5. Effect of drought stress on the development of endosperm protein body and starch granule and starch physicochemical properties in wheat(1) From 12 to 38 DAA, endosperm protein content decreased first and then increased. The amount of protein body accumulation in the sub-aleurone endosperm was higher than that in center endosperm. The amount of protein body accumulation and the protein content were higher in hard wheat cultivar Xumai33 than that in soft wheat cultivar Ningmail3. Drought stess significantly enhanced the sizes and relative areas of protein body in the dorsal and abdominal endosperms. (2) Drought stress respectively resulted in larger size of A-type starch granules at 12 DAA and a higher percentage of B-type starch granules at 18 DAA in endosperm cells. Drought also decreased the percentage of B-type starch granules in Ningmail3 and increased the number of A-type starch granules with hollows on the granule surface in Xumai33. Meanwhile, drought stress decreased the 1000-grain weight, grain total starch, amylose contents and amylose-to-amylopectin ratio. (3) Drought stress had influence on the physicochemical properties of wheat starch. Drought enhanced the swelling power, peak viscosity and trough viscosity of starch in Xumai33 and also enhanced the peak viscosity, trough viscosity and breakdown viscosity of starch in Ningmail3. Drought stress increased the starch hydrolysis degree by a-amylase and decreased the hydrolysis degree by glocoamylase and hydrochloric acid in Ningmail3. However, drought stress had little influence on starch hydrolysis degree in Xumai33. In addition, drought stress also altered the contents of rapidly digestible, slowly digestible, and resistant starches in native, gelatinized, and retrograded starches in two wheat cultivars.Our study revealed the developmental characteristics and the relationships of various wheat caryopsis tissues and elucidated the effect of nitrogen and drought stress on caryopsis development characteristics. The results not only enriched the theoretical knowledge including botany, cell biology, and developmental biology etc. and further deepened the study on the law of wheat caryopsis development and the mechanism of grain quality formation and regulation, but also provided the theoretic guarantees for regulating wheat grain growth, development and quality traits with fertilizer and water management.