Serotonin enrichment of rice endosperm by metabolic engineering

In animals,serotonin is a neurotransmitter and mood regulator.In plants,serotonin functions in energy acquisition,tissue maintenance,delay of senescence,and response to biotic and abiotic stresses.In this study,we examined the effect of serotonin enrichment of rice endosperm on plant growth,endosperm development,and grain quality.To do so,TDCs and T5H were selected as targets for serotonin fortification.Overexpression of TDC1 or TDC3 increased serotonin accumulation relative to overexpression of T5H in rice grain.Transgenic lines of target genes driven by the Gt1 promoter showed better field performance than those driven by the Ubi promoter.Overexpression of T5H showed little effect on plant growth or grain physicochemical quality.In neuronal cell culture assays,serotonin induced neuroprotective action against apoptosis.Breeding of rice cultivars with high serotonin content may be beneficial for health and nutrition.

Quantitative Trait Loci for Grain Chalkiness and Endosperm Transparency Detected in Three Recombinant Inbred Line Populations of Indica Rice

Quantitative trait loci（QTL） for percentage of chalky grain,degree of chalkiness,and endosperm transparency were detected using 3 recombinant inbred line populations derived from crosses between parental lines of commercial three-line hybrids of indica rice.Two of the populations showed great variations on heading date,and the other had a short range of heading date variation.A total of 40 QTLs were detected and fell into 15 regions of 10 chromosomes,of which 5 regions were detected for 1 or more same traits over different populations,2 were detected for different traits in different populations,3 were detected for 2 or all the 3 traits in a single population,and 5 were detected for a single trait in a single population.Most of these QTLs have been reported previously,but a region located on the long arm of chromosome 10 showing significant effects in all the 3 populations has not been reported before.It was shown that a number of gene cloned,including the Wx and Alk for the physiochemical property of rice grain,and GW2,GS3 and GW5 for grain weight and grain size,could have played important roles for the genetic control of grain chalkiness in rice,but there are many more QTLs exerting stable effects for rice chalkiness over different genetic backgrounds.It is worth paying more attentions to these regions which harbor QTL such as the qPCG5.2/qDC5.2/qET5.2 and qPCG10/qDC10/qET10 detected in our study.Our results also showed that the use of segregating populations having high-uniform heading date could greatly increase the efficiency of the identification of QTL responsible for traits that are subjected to great environmental influence.

Effect of nitrogen fertilizer on distribution of starch granules in different regions of wheat endosperm

This study provided visual evidence of a nitrogen effect on starch granules(SGs) in wheat endosperm. Winter wheat(Titicum aestivum L.) cultivar Xumai 30 was cultured under no nitrogen(control) and 240 kg ha-1of nitrogen applied at the booting stage. The number, morphology, and size of Aand B-type SGs in subaleurone of dorsal endosperm(SDE), center of dorsal endosperm(CDE), modified aleurone(MA), subaleurone of ventral endosperm(SVE), and center of ventral endosperm(CVE) were observed under light and electron microscopes.(1) The distribution of SGs in SDE was similar to that in SVE, the distributions of SGs in CDE and CVE were similar, but the distribution of SGs in MA was different from those in the other four endosperm regions. The number of SGs in the five endosperm regions was in the order SDE > CDE > SVE > CVE > MA.(2) Nitrogen increased the number of Aand B-type SGs in SDE and SVE. Nitrogen also increased the number of B-type SGs but decreased the number of A-type SGs in CDE and CVE. Nitrogen decreased the numbers of A-type and B-type SGs in MA. The results suggest that increased N fertilizer application mainly increased the numbers of small SGs and decreased the numbers of large SGs, but that the results varied in different regions of the wheat endosperm.

A Cleaved Amplified Polymorphic Sequence Marker to Detect Variation in Wx Locus Conditioning Translucent Endosperm in Rice

The translucent endosperm trait in a japonica rice variety ＇Kantou 194＇ is controlled by a Wx-mq gene which is allelic to Wx locus by genetic analysis and allelic test. The amylose content analysis showed that an intermediate amylose content between those of glutinous and non-glutinous rice existed in endosperm of homozygous Wx-mq genotype. The slight changes of amylose content in different varieties and F1 grains with an identical Wx-mq genotype might be influenced by dissimilar genetic background. To identify the Wx-mq genotype simply and rapidly, a cleaved amplified polymorphic sequence （CAPS） marker was designed. The result from the molecular detection indicated that it could be used for marker-assisted selection for low amylose content varieties in rice breeding.

Endosperm Development in Autotetraploid Rice

By using the laser scanning confocal microscope and plastic （Leica 7022 historesin embedding kit） semi-thin sectioning technique, comparative studies on the development of endosperm were carried out between autotetraploid and diploid rices. About one third of the ovaries in the autotetraploid showed normal endosperm development as those in the diploid. In these ovaries, one of the polar nuclei would fuse with the sperm nucleus, and the primary endosperm nucleus formed and underwent the first division in 4 hours after pollination; the anticlinal wall began to grow centripetally between the free nuclei starting from the wall ingrowths of the embryo sac near the micropylar end, and some of the phragmoplasts formed transformed into periclinal walls. In addition, some of the cell wall situated in the middle of the endosperm appeared to originate from phragmoplasts, whereas others seemed to develop randomly without the obvious formation of phragmoplasts. Cellulose began to accumulate in the wall of aleurone cell layer at 6 days after pollination. The cellulose wall of the cells of the aleurone cell layer appeared to have completely formed within 7 to 8 days after pollination. On the other hand, about two thirds of the ovaries in the autotetraploid showed abnormality in endosperm development with various types, such as non-fertilization, abnormal fertilization, endosperm development-delay and non-synchronization in the development of cellulose wall of cells of the aleurone layer. These abnormalities usually resulted in decreased seed setting in autotetraploid rice.

Characterization of the imprinting and expression patterns of ZAG2 in maize endosperm and embryo

ZAG2 has been identified as a maternally expressed imprinted gene in maize endosperm.Our study revealed that paternally inherited ZAG2 alleles were imprinted in maize endosperm and embryo at 14 days after pollination(DAP), and consistently imprinted in endosperm at 10, 12, 16, 18, 20, 22, 24, 26, and 28 DAP in reciprocal crosses between B73 and Mo17. ZAG2 alleles were also imprinted in reciprocal crosses between Zheng 58 and Chang7-2 and between Huang C and 178. ZAG2 alleles exhibited differential imprinting in hybrids of 178 × Huang C and B73 × Mo17, while in other hybrids ZAG2 alleles exhibited binary imprinting. The tissue-specific expression pattern of ZAG2 showed that ZAG2 was expressed at a high level in immature ears, suggesting that ZAG2 plays important roles in not only kernel but ear development.

The Effect of High Temperature after Anthesis on Rice Quality and Starch Granule Structure of Endosperm

To ascertain the effect mechanism of high temperature after anthesis on rice quality, the experiment was conducted with two rice lines, the heat-tolerant line 996 and heat-sensitive line 4628, with high temperature and optimal temperature in the growth chamber to investigate the effect of high temperature stress after anthesis on rice appearance quality, milling quality, cooking and eating quality and starch granule structure of endo- sperm. The result showed that milled rice rate, head rice rate, amylose content and gel consistency of both lines decreased under high temperature stress after anthesis, while the ratio of grain length to width, chalky rate, chalkiness, protein content increased. Under high temperature treatments, gelatinization temperature, final viscosity, set back and peak time increased, breakdown decreased, Mg content and K content increased, Mg/K ra- tio decreased. Under same treatment, the extent of rice quality of heat tolerant line 996 affected by high temperature was lower than that of heat sensitive line 4628. Under high temperature stress after anthesis, starch granule arranged untightly, most of starch granules existed in the form of a single starch endosperm, refractive index decreased, transparency reduced, and led to the formation of chalk. Under high temperature stress af- ter anthesis, the increase of protein content, the decrease of Mg/K, the changes of rice RVA profile characteristics and starch granule structure of endosperm could be the main reason for the decrease of rice cooking and eating quality and appearance quality.

Transferring Translucent Endosperm Mutant Gene Wx-mq and Rice Stripe Disease Resistance Gene Stv-bi by Marker-Assisted Selection in Rice (Oryza sativa)

A high-yielding japonica rice variety, Wuyunjing 7, bred in Jiangsu Province, China as a female parent was crossed with a Japanese rice variety Kantou 194, which carries a rice stripe disease resistance gene Stv-b＇ and a translucent endosperm mutant gene Wx-mq. From F2 generations, a sequence characterized amplified region （SCAR） marker tightly linked with Stv-b＇ and a cleaved amplified polymorphic sequence （CAPS） marker for Wx-mq were used for marker-assisted selection. Finally, a new japonica rice line, Ning 9108, with excellent agronomic traits was obtained by multi-generational selection on stripe disease resistance and endosperm appearance. The utilization of the markers from genes related to rice quality and disease resistance was helpful not only for establishing a marker-assisted selection system of high-quality and disease resistance for rice but also for providing important intermediate materials and rapid selection method for good quality, disease resistance and high yield in rice breeding.

Phenotype of Rice Floury Endosperm Mutant flo7 and Fine Mapping of Mutated Gene

The major storage substance in rice endosperm is starch, which accounts for 80% of dry matter weight. In this study, rice mutant flo7, selected from the progeny of Nipponbare＇s tissue culture, displayed floury and opaque endosperm. Compared with its corresponding wild type （WT） Nipponbare, the mutant rio7 produced longer, narrower, thinner and lighter grains. The levels of glucose, fructose and sucrose in the mutant flo7 endosperm were higher than those in the WT endosperm, whereas the protein content was not affected. With respect to both amylose content and gel consistency, the mutant flo7 was lower than WT, but its alkali value was higher. Scanning electron microscopic examinations showed that the endosperm of the mutant flo7 contained irregular, loosely packed and compound starch granules. Genetic analysis indicated that the mutant phenotype was determined by a single recessive nuclear gene The flo7 locus was mapped to a region on the long arm of chromosome 12, within a 95.1 kb interval defined by the markers C2-11 and C5-15. There are 13 open reading frames in the mapping interval. Transcription profiling of the developing grains showed that a number of genes involved in starch synthesis were affected differently in the mutant flo7.

Chalkiness Characters and Scanning Electron Microscope Observation of Rice Grain Endosperm of Japonica Varieties in Southern Henan

Chalkiness characters affect not only the grain appearance,milling,eating and cooking qualities but also the grain nutritional quality in rice,thus it is one of the most important traits in rice. It is very important for us to investigate the relation of the chalkiness formation and the development of endosperm structure and starch granule of different rice varieties. Here,we have investigated the chalkiness characters such as chalkiness rate,chalkiness degree and chalkiness area in 15 japonica rice varieties from southern Henan. Furthermore,the endosperm structure and starch granules of rice grain were also observed with scanning electron microscope. The results showed that the 15 japonica varieties have a significantly linear relationship between the chalkiness rate and chalkiness degree. Among the varieties,the biggest difference is the chalkiness rate,the second is the chalkiness area,and the last is the chalkiness degree. Moreover,there is a certain correlation between the distribution of starch granules,the arrangement of endosperm cells and the occurrence of grain chalkiness in the different rice varieties. For the same variety,the starch granules of chalky and non-chalky grains have obvious difference,while the starch granules from the transparent part of chalky rice and non-chalky rice do not have significant difference. The results would provide useful references for the improvement of grain quality in rice.

Study on Plant Regeneration of Wheat Mature Embryos Under Endosperm-Supported Culture

To reveal the suitability of using mature embryos as an explant source in wheat tissue culture, mature embryos from eight common wheat cultivars （Triticum aestivum L. cv.） were cultured with or without endosperm to test their efficiency of callus induction and plant regeneration. When embryos were cultured together with endosperm （endosperm-supported culture, ES）, the percentage of callus induction was significantly lower than that when embryos were cultured in the absence of endosperm （non-endosperm-supported culture, NES）. This pattern was evident in most genotypes, regardless of whether 2 or 8 mg L^-1 2,4-D was added in the NES culture. However, in ES culture, more induced calli were differentiated into distinct green spots and they further developed into plantlets. Thus, more plants were regenerated in ES culture than in the NES treatment. Most of the eight tested genotypes showed a significant difference in callus induction rate and plantlet regeneration in both ES and NES cultures. In addition, the enzymatic activity of oxalate oxidase in the callus of ES culture condition was obviously higher than that in the callus of NES culture condition, suggesting that the activity of oxalate oxidase may be a parameter for selection of calli with potential for plantlet regeneration. These results indicate that wheat mature embryos are valuable explants for highly efficient callus induction and plant regeneration, if proper treatment and medium are used.

Mitochondrion-targeted PENTATRICOPEPTIDE REPEAT5 is required for cis-splicing of nad4 intron 3 and endosperm development in rice

Endosperm as the storage organ of starch and protein in cereal crops largely determines grain yield and quality.Despite the fact that several pentatricopeptide repeat(PPR)proteins required for endosperm development have been identified in rice,the molecular mechanisms of many P-type PPR proteins in endosperm development remains unclear.Here,we isolated a rice floury endosperm mutant ppr5 that developed small starch grains and an abnormal aleurone layer,accompanied by decreased starch,protein,and amylose contents.Map-based cloning combined with a complementation test demonstrated that PPR5 encodes a P-type PPR protein that is localized to the mitochondria.The mutation in PPR5 caused reduced splicing efficiency of mitochondrial NADH dehydrogenase 4(nad4)gene intron 3 and reduced complex I assembly and activity.Loss of PPR5 function greatly upregulated expression of alternative oxidases(AOXs),reduced ATP production,and affected mitochondrial morphology.We demonstrate that PPR5,as a P-type PPR protein,is required for mitochondrial function and endosperm development by controlling the cis-splicing of mitochondrial nad4 intron 3.

Studies on the Programmed Cell Death in Rice During Starchy Endosperm Development

Morphological variations of the nucleus in starchy endosperm cell were observed by theelectron-transmisson microscope during endosperm development in rice. Along with thedevelopment of the starchy endosperm, the nuclei of the cells showed chromatin condensation,the typical feature of programmed cell death (PCD). The nuclei also showed nucleusdeformation, disruption of nuclear envelope, nucleoplasm leaking into the cytoplasm andnucleus disintegration resulting in nuclear residue formation. From the nucleus deformationto the nucleus disintegration, the morphological changes of the nucleus were orderlyprogressive. This indicated that the cell death of starchy endosperm in rice wasprogrammed cell death. Evans Blue staining observation showed that the cell death wasinitially detected in the central part of starchy endosperm in rice, then expandedoutward. The activities of superoxide dismutase (SOD) and catalase (CAT) in rice starchyendosperm both descended continuously as development progressed. The analysis of DNA ofrice starchy endosperm did not show the presence of DNA laddering. The above resultsshowed that the cell death of starchy endosperm in rice was a special form of PCD.

Identification and Genetic Analysis of a Novel Allelic Variation of Brittle-1 with Endosperm Mutant in Maize

Endosperm mutants are critical to the studies on both starch synthesis and metabolism and genetic improvement of starch quality in maize.In the present study,a novel maize endosperm mutant A0178 of natural variation was used as the experimental material and identified and then characterized.Through phenotypic identification,genetic analysis,main ingredients measurement and embryo rescue,development of genetic mapping population from A0178,the endosperm mutant gene was located.The results showed that the mutant exhibited extremely low germination ability as attributed to the inhibited embryo development,and amounts of sugars were accumulated in the mutant seeds and more sugars content was detected at 23 days after pollination(DAP)in A0178 than B73.Employing genetic linkage analysis,the mutant trait was mapped in the bin 5.04 on chromosome 5.Sequence analysis showed that two sites of base transversion and insertion presented in the protein coding region and non-coding region of the mutant brittle-1(bt1),the adenylate translocator encoding gene involved in the starch synthesis.The single base insertion in the coding region cause frameshift mutation,early termination and lose of function of Brittle-1(BT1).All results suggested that bt1 is a novel allelic gene and the causal gene of this endosperm mutant,providing insights on the mechanism of endosperm formation in maize.

Ultracytochemical Localization and Functional Analysis of ATPase During the Endosperm Development in Oryza sativa L.

Ultracytochemical localization of ATPase during development of rice endosperm was performed using a lead phosphate precipitation technique. The results indicated that, at the coenocyte and cellularization stages, active ATPase was mainly distributed in an embryo sac wall, nucleus, and plasma membrane. At the early stage of development and differentiation, active ATPase was observed in the plasma membrane. At the grain filling stage, ATPase was highly active in the plasma membrane, intercellular space, and plasmodesmata in aleurone, moderately active on the plasma membrane in subaleurone. In starchy endosperm, ATPase was localized in the plasma membrane and degenerated nucleus. ATPase activity also appeared around vacuole and protein body in endosperm cell. The relationships between the ultracytochemical localization of ATPase and its function during the development of rice endosperm were discussed. Overall, ATPase was involved in the process of nutrition absorption and protein synthesis.

Mapping Quantitative Trait Loci Controlling Endosperm Traits with Molecular Marker

Based on the genetic models for triploid endosperm traits and on the methods for mapping diploid quantitative traits loci (QTLs), the genetic constitutions, components of means and genetic variances of QTL controlling endosperm traits under flanking marker genotypes of different generations were presented. From these results, a multiple linear regression method for mapping QTL underlying endosperm traits in cereals was proposed, which used the means of endosperm traits under flanking marker genotypes as a dependent variable, the coefficient of additive effect (d) and dominance effect (h1 and/or h2) of a putative QTL in a given interval as independent variables. This method can work at any position in a genome covered by markers and increase the estimation precision of QTL location and their effects by eliminating the interference of other relative QTLs. This method can also be easily used in other uneven data such as markers and quantitative traits detected or measured in plants and tissues different either in generations or at chromosomal ploidy levels, and in endosperm traits controlled by complicated genetic models considering the effects produced by genotypes of both maternal plants and seeds on them.

The Development of Embryo and Endosperm in Ranalisma rostratum

The embryo and endosperm development in Ranalisma rostratum was studied in this paper.The zygote divides by a transverse wall to form basal and apical cells.The larger basal cell undergoes no further division and becomes the basal suspensor cell.The embryo development conforms to the Caryophyllad type.In the mature seed,the embryo is U shaped and forms the embryonic shoot apex accompanied by one intraseminal leaf.The endosperm development belongs to the Hebolial type.The primary endosperm nucleus (invariably lying in the chalazal part of the embryo sac) divides and forms a large micropylar chamber and a small chalazal chamber.The chalazal endosperm chamber remains uninucleate,and free nuclear divisions occur in the micropylar chamber.Cellularization of the endosperm begins simultaneously from the micropylar and the chalazal ends.The cellularization is related to phragmoplast and cell plate, and does not arise from the free growth wall.Accompanying the embryo development,the endosperm degrades and does not exist in the mature seed.In the proembryo developing stages, the basal suspensor cell and chalazal endosperm cell grow to a considerable size.The nucleus and cytoplasm stain densely.Sometimes, binucleate or trinucleate was observed in both of the two cells.In the laterally concave embryo stage,they begin to degenerate which coincides with the cellularization of the endosperm.They are presumed to participate in the nourishment of the embryo proper in the early stages of embryo development.

Division of Free Nuclei in Rice Endosperm and Its Influencing Factors

The division process of endosperm in rice and its influencing factors were investigated. Total dissection, resin embedding and sectioning under light and transmission electron microscopes were used to observe the division patterns of free nuclei and cytological characters of an endosperm. The various division patterns of free nuclei in rice endosperm included mitosis, amitosis, and the mitosis to amitosis change without an interphase. The division velocity of free nuclei in rice endosperm varied with the internucleus distance. Comparing to a cluster, the division velocity increased when the free nuclei were separated from each other. Moderately higher temperatures also enhanced the proliferation and shortened the division cycle of free nuclei in rice endosperm. To a certain extent, nitrogenous fertilizer applied at the booting stage together with a moderate drought treatment accelerated the division of free nuclei as well.

Proteomic analysis of differentially abundant proteins in the embryo and endosperm of Givotia moluccana (L.) Sreem

Proteome analysis of embryo and endosperm of Givotia moluccana,a promising angiosperm tree species was carried out using two-dimensional electrophoresis combined with matrix-assisted laser desorption/ionization time of flight to identify differentially abundant proteins and their possible functions.The study revealed the presence of 492 protein spots in the embryo and 432 in the endosperm,and the relative abundance of 44 spots varied significantly between the two.Of the 21 functionally characterized protein spots,14.3%were involved in metabolism and energy,28.6%in protein destination and storage,14.3%in defense and stress and 19%in cell growth and division.The embryo-specific protein ATPdependent zinc metalloprotease FtsH is predicted to be involved in chloroplast biogenesis and the endospermspecific protein hydroxyacyl-ACP dehydratase in fatty acid synthesis.These results suggested that the seeds have proteins required for germination and for stress responses.

Analysis of Low Amylose and Processability Fractured Endosperms Derived from Somatic Variation

Recently, improving eating quality and processing properties has become one of the most important objectives in japonica rice breeding programs in Korea. This study was carried out to determine the agronomy and physicochemical characteristics of the opaque endosperm rice "S-21-3-8" regenerated from seed-derived callus culture of a rice cultivar, "Ilpum". S3 generation of opaque endosperm mutants selected from pedigree breeding was used for analysis of agronomic and physicochemical traits. Genetic segregation was observed at the highest frequency among opaque endosperm mutants, being present in 85.7% (12/14 lines) of the entire opaque lines. However, the major agricultural characteristics and grain traits of "S-21-3-8" were similar to those of a donor cultivar, "Ilpum". "S-21-3-8" showed significantly lower (10.6%) amylose than those (17.7%) of "Ilpum" in brown rice, while the protein levels were similar to those of the donor plant. The grain hardness of "S-21-3-8" (1.67 kgf/mm2) was lower than that of "Ilpum" (1.97 kgf/mm2), resulting in a high flour-milling percentage. The loosely packed starch granules of "S-21-3-8" in the opaque endosperm were demonstrated by SEM analysis of cross-sectioned rice grains. The opaque endosperm mutants that were of somaclonal variations in the tissue culture will lead to improved eating quality and processing properties of rice.