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.

高温胁迫下水稻胚乳磷酸化与泛素化蛋白质组学联合分析

水稻胚乳的代谢依赖于多个翻译后修饰信号网络的精确调控,包括磷酸化和泛素化修饰。本研究通过磷酸化与泛素化蛋白质组学的联合分析,发现水稻胚乳中有143个蛋白质同时具有磷酸化和泛素化位点,这为研究磷酸化和泛素化修饰之间的串扰机制提供了组学基础。一方面,磷酸化修饰通过影响泛素-蛋白酶体系统中的关键蛋白来调控泛素化修饰;另一方面,泛素化修饰通过影响蛋白激酶和蛋白磷酸酶来间接调控植物体内的磷酸化信号网络。高温胁迫下,磷酸化和泛素化修饰协同调控酚类化合物的合成通路,并最终影响酚类化合物清除活性氧的能力以及植物在逆境中的抗性。多种淀粉代谢相关酶同时具有磷酸化和泛素化修饰,并且有18对具有不同修饰类型的位点出现在同一蛋白质的相邻位置,揭示了不同修饰之间的串扰机制以及高温胁迫中磷酸化与泛素化修饰对淀粉代谢的协同调节作用。

营养胁迫对小麦幼苗生长和生理特性的影响

为了探究小麦幼苗生长潜力及其对营养胁迫的适应能力,通过沙培试验,设置了正常生长植株和种子营养物质缺失(仅去除胚乳、仅去除根系、胚乳和根系均去除)处理,考察了小麦苗期叶龄、生长速率、株高、根系、叶绿素含量(SPAD)、叶面积、干物质积累、生理特性的变化。结果显示,萌发36 h的小麦幼苗在营养缺失处理下均已具备独自存活能力。与未去除胚乳(正常植株和仅去除根系)的小麦幼苗相比,去除胚乳(仅去除胚乳以及胚乳和根系均去除)的小麦幼苗发育缓慢,需要更多时间才能达到与正常小麦相同的生长阶段,且在两叶一心时生长速率、株高、根长、SPAD、总叶面积和干重显著下降,长势明显较弱,可溶性蛋白、脯氨酸和丙二醛含量一直维持在较低的水平。与正常生长植株相比,仅去除根系的小麦幼苗能够快速适应营养胁迫,在两叶一心时生长速率、株高、根长和干重与正常植株基本一致,可溶性蛋白和脯氨酸含量显著增加,丙二醛含量显著降低。由此可见,苗期小麦种子营养物质缺失条件下幼苗生长迟缓,适应能力弱,影响小麦后期的生长和发育。

玉米籽粒胚乳细胞相关性状的杂种优势研究

为了解析玉米粒重性状杂种优势的产生原因,对籽粒胚乳细胞数目、细胞大小开展了研究。以玉米品种‘郑单958’、‘先玉335’及其亲本为试验材料,分析了杂交种与亲本在单籽粒重量、单籽粒胚乳细胞数目及胚乳细胞面积的杂种优势关系。结果表明,‘先玉335’和‘郑单958’胚乳细胞数目的中亲优势分别为40.81%和36.03%,超亲优势分别为33.09%和23.82%;百粒重的中亲优势分别为27.22%和28.89%,超亲优势分别为19.72%和18.34%;而胚乳细胞面积的中亲优势分别为-44.42%和-24.79%,超亲优势分别为-45.75%和-37.30%。单籽粒胚乳细胞数目与粒重呈极显著正相关,相关系数为0.796。综上所述,玉米杂交种籽粒的胚乳细胞数目多于亲本是粒重杂种优势产生的重要原因,选育籽粒胚乳细胞数目多的自交系进行杂交组配利于提高杂交种的粒重。

毛竹种子形成过程中形态解剖学特征

为揭示毛竹(Phyllostachys edulis)种子生长过程中胚、胚乳、果皮及种皮的发育规律,以桂林海洋山一带的开花毛竹为材料,采集并固定不同时期的开花毛竹种子,使用石蜡制片法制片,显微镜观察胚、胚乳、果皮与种皮的结构变化。结果表明:(1)毛竹花后1 d完成受精并形成合子,合子休眠时长约为5 d。经过原胚阶段、胚芽鞘阶段、幼胚生长阶段及成熟胚阶段,花后40 d的胚发育基本成熟,其发育类型为禾本型。(2)胚乳发育早于胚的发育,其发育类型为核型胚乳,历经游离核、细胞化、细胞分化及成熟4个阶段。在细胞分化阶段胚乳细胞分化形成淀粉胚乳细胞以及糊粉层细胞,淀粉胚乳细胞主要积累淀粉粒,糊粉层细胞主要积累矿质元素、脂类及蛋白质等。(3)花后1 d的果皮细胞及珠被细胞形状规则、内含物丰富、结构完整;花后10~20 d,内、外果皮及珠被细胞层数递减,形状发生改变,中果皮细胞开始出现淀粉粒;花后20~60 d,随着胚乳细胞营养物质的积累及体积的增大,向外产生机械压力,中果皮细胞逐步消解仅剩残留的细胞壁;外果皮细胞呈长条形,细胞壁加厚,与残留的中果皮细胞壁组成保护结构;皮层在种子发育过程中主要起到合成、运输营养物质以及保护胚和胚乳发育的作用。该研究结果为完善毛竹生殖生物学相关内容以及了解竹类植物胚和胚乳的发育提供了理论依据。

Ultrastructural Features of Nucleus Degradation During Programmed Cell Death of Starchy Endosperm Cells in Rice

Ultrastructural features of nucleus degradation during programmed cell death (PCD) of starchy endosperm cells in rice ( Oryza sativa L.) were observed using transmission electron microscopy. Several distinct morphological features of PCD have been found in the developing starchy endosperm cells, e.g. nucleus deformation, chromatin condensation, nuclear envelope disruption, and nuclear matrix leakage. DNA ladder displayed a smear of large DNA fragments from nucleus and evident bands of small DNA fragments (140-180 bp) from both nucleus and cytoplasm. In contrast with the rapid nucleus degradation, cell organelles in cytoplasm, such as rough endoplasmic reticulum, amyloplast, and mitochondrion, maintained their metabolic functions for a longer time. Seed reserves were continually synthesized and accumulated in the starchy endosperm cells despite the nucleus degradation during the PCD process. These results suggest that starchy endosperm cells remain active during reserve material synthesis and accumulation in the PCD process. The specific relationships between nucleus and cytoplasm in the developing endosperm cells and the morphological changes of nucleus in the endosperm PCD process were also discussed.

Interval Mapping of Quantitative Trait Loci Underlying Triploid Endosperm Traits Using F_3 Seeds

A statistical method for mapping quantitative trait loci （QTLs） underlying endosperm traits is proposed. The method is based on a genetic model containing both the direct effects and maternal effects of an endosperm QTL and on an experimental design termed two-stage hierarchical design, in which the trait information is obtained from F3 endosperms and molecular marker information is obtained from F2 plants and F3 embryos （plants）. Results of computer simulations indicate that the method can efficiently map endosperm QTLs and precisely estimate both the direct and maternal effects of endosperm QTLs when the sample size is sufficiently large.

H5N1亚型禽流感病毒HA蛋白在水稻胚乳中的表达及其纯化

为制备H5N1亚型禽流感病毒HA蛋白并评估其免疫原性,利用水稻表达系统表达H5N1亚型禽流感病毒重组HA蛋白。首先构建了重组植物表达载体pCAMBIA1300-HA,该载体具有GT13特有启动子、信号肽SP和终止子,有利于外源基因在水稻中表达。之后将重组质粒pCAMBIA1300-HA通过电转化法导入根癌农杆菌EHA105中,筛选出阳性菌落并侵染水稻愈伤组织。经过暗培养、潮霉素筛选、分化、生根、成苗,采用CTAB法提取水稻叶片DNA,PCR鉴定结果显示,HA基因已插入到水稻基因组中,重组HA基因大小为4660 bp。将阳性植株移植到大田中,4个月后收获水稻种子,提取水稻胚乳蛋白,Western blot鉴定结果表明,HA蛋白在水稻胚乳中成功表达。重组HA蛋白通过Q阴离子层析、疏水层析和凝胶过滤层析三步分离纯化,其纯度达到90%以上。最后将纯化后的重组HA蛋白与ISA 50V佐剂混合并乳化制成疫苗,免疫小鼠,小鼠血清具有较高的特异性抗体水平,表明重组HA蛋白免疫原性较好。综上,成功构建了H5N1亚型禽流感病毒HA重组蛋白的水稻表达系统,并分离纯化获得了高纯度和免疫原性良好的重组HA蛋白。

In Vitro Development of Tobacco Primary Endosperm Cells in Microculture

用酶解_研磨法分离出烟草 (NicotianatabacumL .)受精后胚囊和初生胚乳细胞进行微室饲养培养。培养基为Km8p附加各种其他成分 ,饲养细胞为分裂旺盛的烟草叶肉原生质体 ,在 2 5℃下静止暗培养。培养 3d后 ,初生胚乳细胞开始第一次分裂 ,继续分裂至 14d时形成大的细胞团。首次报道了双子叶植物初生胚乳细胞的离体发育。

含热带种质玉米自交系ZOL-1胚乳发育的转录组研究

【目的】研究含热带种质玉米自交系胚乳发育分子机制及相关调控网络。【方法】通过高通量RNA-seq测序技术,对热带血缘玉米自交系ZOL-1在授粉后5、10、15、20、25、30 d 6个时期的胚乳转录组进行比较,得到的差异表达基因进行KEGG与GO功能注释,并对FPKM平均值大于1的基因进行时间序列分析。【结果】玉米自交系ZOL-1胚乳发育在授粉后25~30 d时籽粒长、宽、厚增长速率最快,增长率分别达到15.9%、16.1%和10.2%。经过转录组测序和同参考基因组比对,过滤后reads占比至少为83.99%,测序质量和组装效果均较好。按照连续比较系统方法,在10 d_VS_5 d、15 d_VS_10 d、20 d_VS_15 d、25 d_VS_20 d和30 d_VS_25 d分别检测到7129、5252、4254、775和312个差异表达基因。GO功能注释表明,差异表达基因主要分布于细胞分化过程、代谢过程、催化活性和细胞器等条目中。KEGG功能富集分析显示,差异表达基因主要富集在能量代谢、碳水化合物代谢、碳代谢、氨基酸代谢、信号转导等与胚乳发育相关的通路。通过对FPKM平均值大于1的基因进行时间序列分析,19554个基因共划分为10个Clusters,并在Cluster 7鉴定出8个淀粉合成酶相关基因,它们的表达量在授粉后15 d时开始显著提升,此后保持较高表达水平。【结论】玉米自交系胚乳发育可能主要受下调基因表达调控,在授粉后15 d前后胚乳的发育模式发生变化,转录组有重新编程的迹象,同时挖掘到玉米淀粉生物合成相关基因8个,有利于研究胚乳发育的分子调控机理。

Introgression of lac6/tl1/du13 improves the palatability of japonica rice

Amylose content(AC)is a crucial determinant of the eating and cooking quality(ECQ)of rice,with low AC varieties exhibiting a softer texture and greater stickiness-attributes that enhance palatability and are desirable in specific culinary contexts.To harness these traits,significant efforts have been made to manipulate AC to improve rice ECQ.Our research utilized the MutMap+approach to identify LAC6/TL1,a gene that is an allele of Du13,responsible for low AC.LAC6 encodes a C2H2 zinc finger protein,which specifically increases the splicing efficiency of the Wxb allele without affecting the Wxa allele.Functional studies of LAC6 revealed that its proper integration could rectify the undesirable AC phenotype,whereas mutations within this gene led to reduced AC and were associated with shorter grain length and decreased thousand-grain weight.Despite these drawbacks,such mutations positively impact rice palatability,presenting a trade-off between grain size and eating quality.To address the challenges posed by the reduced grain weight associated with LAC6 mutations,we developed a specific molecular marker for LAC6,which has been effectively used in breeding programs to select lac6/tl1/du13 homozygous individuals with larger grain size.Our findings demonstrate that the“small grain”trait associated with lac6/tl1/du13 can be effectively mitigated through combined phenotype-based and marker-assisted selection.This study highlights the potential of lac6/tl1/du13 as a valuable gene for breeding novel,high-quality soft rice varieties through targeted breeding strategies.

Study on FTIR Spectra of Corn Germs and Endosperms of Three Different Colors Combining with Cluster Analysis

[Objective] This research aimed to study the FTIR spectra of corn germs and endosperms so as to provide a scientific way for identifying corn of different types. [Method] The corn germs and endosperms of three types were studied by using Fourier transform infrared spectroscopy（FTIR） technology, combined with cluster analysis. [Result] The overall characteristics of original FTIR spectra were basically similar within the range of 700-1 800 cm^-1. The FTIR spectra were mainly composed by the absorption peaks of polysaccharides, proteins and lipids. Within the wavelength range of 700-1 800 cm^-1, there were only tiny differences in original FTIR spectra among the corn germs and endosperms of three different types. The spectra were then processed by using first derivative and second derivative. The second derivative spectra were used for hierarchical cluster analysis（HCA）. The results showed that with the wavelength range of 700-1 800 cm^-1, the second derivative spectra of the 52 samples could be better clustered according to the tree types and corn germ and corn endosperm. The clustering correct rate reached 96.1%.[Conclusion] FTIR technology, combined with cluster analysis, can be used to identify different types of corn germs and endosperms, and it is characterized by convenience and rapidness.

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.

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.

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.

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.

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.

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.