Effects of Nitrogen Application Time on Caryopsis Development and Grain Quality of Rice Variety Yangdao 6

A pot experiment was conducted to study the effects of different nitrogen application time （during the tillering or the booting stages） with the same nitrogen rates on the caryopsis development and grain quality of rice variety Yangdao 6. The increased nitrogen fertilizer （urea）, especially applied during the booting stage, could evidently increase the milled rice rate, head rice rate and protein content in rice grains compared with the control （no nitrogen application）, and decrease chalky grain rate and amylose content. Moreover, the increased nitrogen fertilizer significantly affected the caryopsis development and enhanced the grain weight when nitrogen applied during the tillering and the booting stages, especially during the booting stage. During caryopsis development the increased nitrogen fertilizer applied during the tillering and booting stages could obviously decrease the total starch and amylose contents, but not obviously for the amylopectin content in rice grain. Increased topdressing of nitrogen fertilizer, especially applied during the booting stage, had significant effect on the development and structures of amyloplasts and proteinoplasts. That is, it could change the distribution, number and shape of amyloplasts and proteinoplasts in the endosperm cells especially in grain abdomen, Compared with the control the arrangements of amyloplasts and proteinoplasts were closer, with more numbers, higher density and less interspaces each ohter. Furthermore, most amyloplasts showed polyhedron under the increased nitrogen fertilizer level.

Effects of Nitrogen Fertilizer Treatments on Caryopsis Filling and Respiratory Rate of Rice

An experiment was conducted to study the effects of nitrogen （N） rate and application time on grain filling and respiratory trait of caryopsis in two rice varieties, IR36 and Dali. The treatments were consisted of no N application topdressing at both tillering and booting stages （CK）, 6 g/pot of N topdressing at the tillering stage and 2 g/pot of N topdressing at the booting stage, 2 g/pot of N topdressing at the tillering stage and 6 g/pot of N topdressing at the booting stage. The results showed that the proper utilization of N fertilizer can be helpful to maintain the higher water content, higher respiratory rate and higher dehydrogenase activity of rice caryopsis in late filling phase, and prolong the course for filling and maintaining higher respiratory rate and dehydrogenase activity of rice caryopsis. More N application at booting was more effective compared to more N application at tillering.

Structural and Histochemical Characterization of Developing Rice Caryopsis

The development of pericarp, seed coat, starchy endosperm and aleurone of the rice caryopsis was investigated, histochemically and structurally, from the time of flowering to maturity. The results showed that during its growth, the maximum length of the caryopsis was attained first, followed by width and then thickness. Histochemical examination of the caryopsis showed that starch was mainly accumulated in the endosperm, but the endosperm showed no metabolic activity, while embryo and pericarp contained a few starch grains, and embryo and aleurone were strongly active. Aleuronic cells contained many aleurone grains and spherosomes, and aleurone in the dorsal region developed earlier and contained more layers of cells. Amyloplasts in endosperm contained many starch granules and were spherical at early stages but polyhedric at late stages. The protein bodies appeared later than amyloplasts, and the number of protein bodies in subaleurone was greater than those in the starchy endosperm. The white-belly portion of endosperm might be relative to the status of amyloplast development.

OsPEX1,a leucine-rich repeat extensin protein,functions in the regulation of caryopsis development and quality in rice

Rice caryopses are enclosed by outer glumes.The size and dimension of the outer glume are the main determinants of caryopsis size.However,it is unclear whether caryopsis development is completely dependent on the size of the glume,or whether it can grow and expand autonomously despite the constraint of glume enclosure.We report the identification of a mutant line that produces normal-sized glumes with smaller mature caryopses that do not fill the entire glume cavity.The caryopsis phenotype in the pex1 mutant is caused by a reduction in cell size.Os PEX1,a leucine-rich repeat extensin gene,was highly expressed in the developing caryopsis.Overexpression of Os PEX1 driven by a constitutive promoter recapitulated the mutant phenotype,showing that the small-caryopsis phenotype is caused by overexpression of the Os PEX1 gene.Free amino acids,including several essential amino acids,and crude protein were increased in pex1 relative to the wild type,endowing pex1 with improved nutritional quality.Our results suggest that caryopsis development can be genetically uncoupled from maternally controlled glume development and that Os PEX1 might be a new resource for improving nutritional quality of rice cultivars.

The Association of Chalazal Cell Ultrastructure in Caryopsis and Grain Filling in Wheat

Using three wheat cultivars (Triticum aestivum L. ) , Een1, Huamai8 and 95A-10, which exhibit different grain filling characters. the relationship of ultrastructure of the chalazal cells in wheat caryop-sis and grain filling was studied. The results indicate that the lipoid and polysaccharide deposited in vacuoles of the chalazal cells have no obvious inhibitory actions to the grain filling. It appears that a direct relationship exists between the proceeding and end time of grain filling and the amount and appearing time of the lipoid, within lignin and suberin deposited on the inner wall of chalazal cells, and between grain filling rate and cultivars characteristic. The appearing time of the lipoid, within lignin and suberin deposited on the inner wall of chalazal cells in cultivar with large grain (Eenl and 95A-10) was later than that in cultivar with small grain (Huamai8), and the amount of deposits in the former was less than that in the latter. The late development of caryopsis and low rate of grain filling account for the wrinkled grain of 95A-10. The transport of assimilates in chalazal cells coexists with symplastic and apoplastic pathways, but symplastic pathway plays a major role after 24 days of flowering.

Caryopsis Development and Main Quality Characteristics in Different indica Rice Varieties

A comparison on the caryopsis development, rice quality characteristics and the size, shape, structure of the endosperm amyloplasts （starch granules） between two indica rice varieties was made, The main quality traits in Yangdao 6 were better than those in Xiangzaoxian 33; In the early period after fertilization the dry matter in Yangdao 6 was accumulated more slowly than that in Xiangzaoxian 33 but faster in the later period, and the starch was accumulated strongly in the later period; There were two kinds of amyloplasts： single and compound amyloplasts, being 4.4 um and 9.5 um in diameter on average with the range of 2.4-8.0 um and 5.7-19.5 um, respectively. In the case of Xiangzaoxian 33, most of the single amyloplasts were elliptic or round with loose arrangement and great difference in size, and the coefficient of variation was high. While in the Yangdao 6, most of the amyloplasts were single, well developed, polyhedral, crystalline and compactly arranged, and the coefficient of variation was low. The amyloplasts in the dorsal region of endosperm were developed better than those in the ventral and central regions. The chalkiness in the endosperm resulted from badly-developed and loose-arranged starch granules, which was closely relevant to the transport of filling materials.

Development and Substance Accumulation of Caryopsis in Transgenic Rice with Antisense Wx Gene

The development and substance accumulation of rice caryopsis were studied by using the transgenic japonica and indica rice with antisense Wx gene. The weight of caryopses in transgenic rice was lower than that in non-transgenic one, and the reduction in weight was significantly correlated to the reduction in amylose content. In caryopsis of transgenic rice, the number of endosperm cells was less than that in caryopsis of non-transgenic one, but the proliferation speed was considerably higher during the first six days after flowering （DAF）. During the first nine DAF, the soluble sugar content of transgenic rice caryopsis was less than that of the non-transgenic one, but the situation was reverse after nine DAF. Moreover, the total starch content also declined with the decrease in amylose content of transgenic rice caryopsis, while the amylopectin content increased accordingly. Therefore, the composition of starch in caryopsis also changed, but it did not affect the accumulation of protein in transgenic rice caryopsis.

GRAIN INCOMPLETE FILLING 2 regulates grain filling and starch synthesis during rice caryopsis development

Rice grain filling determines grain weight, final yield and grain quality. Here, a rice defective grain filling mutant, gif2, was identified. Grains ofgif2 showed a slower filling rate and a significant lower final grain weight and yield compared to wild-type. The starch content in gilt2 was noticeably decreased and its physicochemical properties were also altered. Moreover, gif2 endosperm cells showed obvious defects in compound granule formation. Posi- tional cloning identified GIF2 to encode an ADP-glucose pyrophosphorylase （AGP） large subunit, AGPL2; consequently, AGP enzyme activity in gif2 endosperms was remarkably decreased. GIF2 is mainly expressed in developing grains and the coded protein localizes in the cytosol. Yeast two hybrid assay showed that GIF2 interacted with AGP small subunits OsAGPS% OsAGPS2a and OsAGPS2b. Transcript levels for granule-bound starch synthase, starch synthase, starch branching enzyme and starch debranching enzyme were distinctly elevated in gif2 grains. In addition, the level of nucleotide diversity of the GIF2 locus was extremely low in both cultivated and wild rice. All of these results suggest that GIF2 plays important roles in the regulation of grain filling and starch biosynthesis during caryopsis development, and that it has been preserved during selection throughout domestication of modern rice.

Rice caryopsis development Ⅱ: Dynamic changes in the endosperm

The rice endosperm plays crucial roles in nourishing the embryo during embryogenesis and seed germination. Although previous studies have provided the general information about rice endosperm, a systematic investigation throughout the entire endosperm developmental process is still lacking. In this study, we examined in detail rice endosperm development on a daily basis throughout the 3o-day period of post-fertilization development. We observed that coenocytic nuclear division occurred in the first 2 days after pollination （DAP）, cellularization occurred between 3 and 5 DAP, differentiation of the aleurone and starchy endosperm occurred between 6 and 9 DAP, and accumulation of storage products occurred concurrently with the aleurone/starchy endosperm differentiation from 6 DAP onwards and was accomplished by 21 DAP. Changes in cytoplasmic membrane permeability, possibly caused by programmed cell death, were observed in the central region of the starchy endosperm at 8 DAP, and expanded to the whole starchy endosperm at 21 DAP when the aleurone is the only living component in the endosperm. Further, we observed that a distinct multi-layered dorsal aleurone formed near the dorsal vascular bundle, while the single- or occasionally two-cell layered aleurone was located in the lateral and ventral positions of endosperm. Our results provide in detail the dynamic changes in mitotic divisions, cellularization, cell differentiation, storage product accumulation, and programmed cell death that occur during rice endosperm development.

A Comparative Study on the Role of Cytokinins in Caryopsis Development in the Maize miniature1 Seed Mutant and Its Wild Type

We report here on a comparative developmental profile of plant hormone cytokinins in relation to cell size, cell number and endoreduplicaUon in developing maize caryopsis of a cell wall invertase-deficient miniature1 （mn1） seed mutant and its wild type, Mn1, genotype. Both genotypes showed extremely high levels of total cytokinins during the very early stages of development, followed by a marked and genotype specific reduction. While the decrease of cytokinins in Mn1 was associated with their deactivation by 9-glucosylation, the absolute and the relative part of active cytokinin forms was higher in the mutant. During the exponential growth phase of endosperm between 6 d after pollination and 9 d after pollination, the mean cell doubling time, the absolute growth rate and the level of endoreduplication were similar in the two genotypes. However, the entire duration of growth was longer in Mnl compared with mnl, resulting in a significantly higher cell number in the Mnl endosperm. These data correlate with the previously reported peak levels of the Mn1-encoded cell wall invertase-2 （INCW2） at 12 d after pollination in the Mn1 endosperm. A model showing possible crosstalk among cytokinins, cell cycle and cell wall invertase as causal to increased cell number and sink strength of the Mn1 developing endosperm is discussed.

Rice caryopsis development Ⅰ: Dynamic changes in different cell layers

Rice caryopsis as one of the most important food sources for humans has a complex structure that is composed of maternal tissues including the pericarp and testa and filial tissues including the endosperm and embryo. Although rice caryopsis studies have been conducted previously, a system- atic characterization throughout the entire developmental process is still lacking. In this study, detailed morphological examinations of caryopses were made during the entire 30- day developmental process. We observed some rapid changes in cell differentiation events and cataloged how cellular degeneration processes occurred in maternal tissues. The differentiations of tube cells and cross cells were achieved by 9 days after pollination （DAP）. In the testa, the outer integument was degenerated by 3 DAP, while the outer layer of the inner integument degenerated by 7 DAP. In the nucellus, all tissues with the exception of the nucellar projection and the nucellar epidermis degenerated in the first 5 DAP. By 21 DAP, all maternal tissues, including vascular bundles, the nucellar projection and the nucellar epidermal cells were degenerated. In summary, this study provides a complete atlas of the dynamic changes in cell differentiation and degeneration for individual maternal cell layers of rice caryopsis.

Evaluation of Protocols Used in 2-D Electrophoresis for Proteome Analysis of Young Rice Caryopsis

In order to obtain a high-resolution electrophorogram of rice young panicle proteome, we evaluated various protocols commonly used in two-dimensional （2D） polyacrylamide gel electrophoresis （PAGE） of proteins, including gel staining protocol, pH range of immobilized pH gradient （IPG） strips and sample loading quantity. Results showed that a silver staining protocol using sensitized solution containing glacial acetic acid, sodium acetate and sodium thiosulfate （reported by Heukeshoven and Dernick in 1988） and a Coomassie Brilliant Blue staining method using solution containing G-250, ammonium sulfate and phosphoric acid （reported by Pink et al in 2010） demonstrated the superior staining effect. In addition, we also showed that higher resolution was achieved when IPG gel strip with pH range of 5-8 was used, compared to that with pH range of 4-7. Finally, the optimal loading quantity was determined as 130 ug using the 17 cm-long nonlinear IPG strip with pH 5-8 in combination with the silver nitrate staining protocol. The evaluated results would be helpful in proteome analysis of young rice caryopsis.

Responses of Caryopsis Germination, Seedling Emergence, and Development to Sand Water Content of Agropyron cristatum （L.） Gaertn. and Bromus inermis Leyss.

Responses of caryopsis germination, seedling emergence, and development of Agropyron cristatum （L.） Gaertn. （Gramineae） and Bromus inermis Leyss. （Gramineae）, two dominant perennial grasses in the Otindag Sandland of China, to different sand water content （SWC; 1%, 2%, 3%, 4%, 6%, 8%, 12%, 16%, and 20%） were studied comparatively. The results showed that the germination responses of the two grasses to SWC were similar （i.e. caryopses could not germinate when the SWC was below 3%; at SWC ranging from 3% to 12%, the higher the SWC, the higher the germination percentage; and at a SWC of 12%-20%, germination reached similarly high percentages）. At a sand burial depth of 0.5 cm, the threshold of SWC for seedling emergence was 6% forA. cristatum and 8% forB. inermis; at 12%-20% SWC, the seedling emergence of both species reached similarly high percentages. The seedling growth responses of these two species to SWC gradients were different. For A. cristatum, the biomass of seedlings increased with SWC from 6% to 12%, and decreased with SWC from 12% to 20%. For B. inermis, the biomass of seedlings always increased with SWC from 8% to 20%. The results also showed that the seedlings of both species allocated more biomass to the roots with decreases in SWC. The SWC changes from April to October in natural microhabitats of both species suggested that the SWC may play an important role in caryopsis germination, seedling emergence, and the growth characteristics of the two grasses. The responses of caryopsis germination, seedling emergence, and the growth characteristics of these two species to SWC may determine their distribution pattems in the Otindag Sandland.

Transcriptome Analysis of High-Temperature Stress in Developing Barley Caryopses： Early Stress Responses and Effects on Storage Compound Biosynthesis

High-temperature stress, like any abiotic stress, impairs the physiology and development of plants, including the stages of seed setting and ripening. We used the Affymetrix 22K Barley1 GeneChip microarray to investigate the response of developing barley （Hordeum vulgare） seeds, termed caryopses, after 0.5, 3, and 6 h of heat stress exposure; 958 induced and 1122 repressed genes exhibited spatial and temporal expression patterns that provide a detailed insight into the caryopses＇ early heat stress responses. Down-regulation of genes related to storage compound biosynthesis and cell growth provides evidence for a rapid impairment of the caryopsis＇ development. Increased levels of sugars and amino acids were indicative for both production of compatible solutes and feedback-induced accumulation of substrates for storage compound biosynthesis. Metadata analysis identified embryo and endosperm as primary locations of heat stress responses, indicating a strong impact of short-term heat stress on central developmental functions of the caryopsis. A comparison with heat stress responses in Arabidopsis shoots and drought stress responses in barley caryopses identified both conserved and presumably heat- and caryopsis-specific stress-responsive genes. Summarized, our data provide an important basis for further investigation of gene functions in order to aid an improved heat tolerance and reduced losses of yield in barley as a model for cereal crops.