水稻胚乳组织的结构观察

【目的】阐明水稻糊粉层细胞、亚糊粉层细胞与中心胚乳贮藏细胞的结构特性。【方法】采用光镜、透射电镜与扫描电镜对水稻胚乳组织进行观察研究。【结果】糊粉层细胞分化过程中,大液泡变成小体积蛋白贮存液泡,蛋白贮存液泡又转变成糊粉粒。颖果背部比腹部有更多层糊粉层,但背部糊粉层细胞内糊粉粒的形成与积累速度却较慢。亚糊粉层细胞起初含有一些脂质体,后来脂质体消失,而其内部淀粉体与蛋白体逐渐增多。中心胚乳贮藏细胞含有淀粉体与蛋白体,蛋白体以液泡型蛋白体为主,它们可以相互融合而变大。中心胚乳贮藏细胞内的淀粉积累速度明显快于亚糊粉层细胞内的。成熟颖果的中心胚乳贮藏细胞内淀粉体最为密集,背部和侧部的亚糊粉层细胞内淀粉体排列较疏松,腹部的亚糊粉层细胞内淀粉体最为稀疏。【结论】水稻颖果背部与腹部的糊粉层细胞和亚糊粉层细胞的结构差异可能与养分吸收与转运有关;中心胚乳贮藏细胞内淀粉体发育速度快于亚糊粉层细胞。     

Protein content,distribution and retention during milling of brown rice

Brown rice of IR36 and IR42 check samples from the 1982 dry and wet season yield trials showed good correlation of milled-rice protein and brown-rice protein (r=0.97**,n=40) with 10% bran-polish removal by an emery abrasive mill. Among selected grain samples of both varieties, 80 to 86% of brown-rice protein was retained in the milled rice with both friction and abrasive mills. Stereological morphometry of 1-µm-thick sections showed that endosperm storage protein decreases in amount with increasing distance from the aleurone layer. A similar protein gradient was found in both average-protein and high-protein samples. Milling removed all of the pericarp, seed coat and nucellus, and virtually all of the aleurone layer and embryo, but removed very little of the nonaleurone endosperm, except from the lateral ridges.     

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.     

小麦胚乳发育过程中贮藏蛋白的积累和蛋白体的形成

为给小麦品质育种提供参考,采用光学和电子显微镜技术系统观察了小麦品种扬麦12号胚乳发育过程中贮藏蛋白的积累和蛋白体的形成.结果表明,花后8 d的淀粉胚乳细胞开始出现贮藏蛋白颗粒.贮藏蛋白在粗内质网上合成后,有些经过高尔基体形成浓密小泡,有些直接在粗内质网腔中积累,脱离内质网形成蛋白体;浓密小泡和蛋白体进入蛋白贮藏液泡(PSV).早期的蛋白体较小,呈球状;随着胚乳发育,蛋白体可以相互融合,扩大体积.后期的蛋白体相互融合形成蛋白质基质,单个蛋白体不再存在;接近成熟期的胚乳,胚乳细胞被贮藏物质淀粉粒和蛋白质基质所充实,蛋白质基质分布在淀粉粒之间的空隙内.试验结果说明,贮藏蛋白从内质网到PSV的转运有两种途径:依赖高尔基体途径和内质网衍生出蛋白体途径.     

The Endosperm Morphology of Rice and its Wild Relatives as Observed by Scanning Electron Microscopy

While cultivated rice, Oryza sativa, is arguably the world’s most important cereal crop, there is little comparative morphological information available for the grain of rice wild relatives. In this study, the endosperm of 16 rice wild relatives were compared to O. sativa subspecies indica and O. sativa subspecies japonica using scanning electron microscopy. Although the aleurone, starch granules, protein bodies and endosperm cell shapes of the cultivated and non-cultivated species were similar, several differences were observed. The starch granules of some wild species had internal channels that have not been reported in cultivated rice. Oryza longiglumis, Microlaena stipoides and Potamophila parviflora, had an aleurone that was only one-cell thick in contrast to the multiple cell layers observed in the aleurone of the remaining Oryza species. The similarity of the endosperm morphology of undomesticated species with cultivated rice suggests that some wild species may have similar functional properties. Obtaining a better understanding of the wild rice species grain ultrastructure will assist in identifying potential opportunities for development of these wild species as new cultivated crops or for their inclusion in plant improvement programmes.     

Specific expression of a sesame storage protein in transgenic rice bran

Rice oil bodies enclosed by unique structural proteins, oleosins, are found in the embryo and the aleurone layer, but not the starchy endosperm where starch and storage proteins are accumulated. To examine oleosin promoter specificity, a sesame storage protein, 2S albumin, was expressed in transgenic rice seeds under the control of a rice oleosin promoter. In all transgenic rice seeds, the sesame 2S albumin was found exclusively in the bran fraction after milling. Immunological staining revealed that the sesame 2S albumin was also located in the embryo and the outermost cells of the starchy endosperm. Furthermore, immunogold labeling showed that the transgenic 2S albumin was deposited in both type-I and type-II protein bodies of the outermost cells of the endosperm as well as in the type-II protein bodies of the embryo. The methionine and cysteine contents in the bran from four homozygous transgenic lines were elevated by 24–38 and 50–62%, respectively, compared with those of wild-type plants. The results suggest that the rice oleosin promoter is bran-specific and could be used to add value to rice bran, an abundant by-product of rice polishing, by genetic engineering.     

水稻胚乳细胞发育的结构观察及其矿质元素分析

 为了探明水稻胚乳发育的过程和糊粉层形成的机理, 用光学显微镜和电子显微镜观察了水稻糊粉层细胞和内胚乳细胞在颖果发育过程中的结构变化,用能谱仪分析了胚乳细胞中元素的种类和相对含量。结果表明, 糊粉层细胞是由胚乳表层细胞转化而来的。糊粉层细胞中的P、K、Mg和Ca等矿质元素含量要明显高于内胚乳细胞。发育初期糊粉层细胞中富含线粒体、圆球体和小液泡;发育中后期小液泡积累蛋白质和矿质元素而形成糊粉粒。在发育中后期,内胚乳细胞随着细胞内淀粉体的充实,细胞核发生形变而衰亡;而糊粉层细胞的核在发育过程中不消亡。糊粉层的形成与表层细胞积聚矿质和脂类等“灌浆废物”(指非内胚乳细胞的贮藏物)有关。因而,转运灌浆物质多的胚乳背部,其糊粉层细胞的层数要比腹部和侧部多。谷物胚乳发育分为游离核期、细胞化期、分化期和成熟期四个时期。     

Sesame oleosin and prepro-2S albumin expressed as a fusion polypeptide in transgenic rice were split, processed and separately assembled into oil bodies and protein bodies

A recombinant polypeptide containing the precursor protein of a sesame storage protein, 2S albumin, fused to the C-terminus of a sesame oleosin was expressed in transgenic rice seeds under the control of a rice glutelin promoter. The recombinant polypeptide of 32 kDa, equivalent to the resultant molecular mass of sesame oleosin (15 kDa) and prepro-2S albumin (17 kDa), was detected in the endoplasmic reticulum fraction of maturing transgenic rice seeds, but not in the purified oil bodies or the soluble extract of transgenic seeds. However, sesame oleosin presumably fused with a 2 kDa C-terminal appendix originating from the signal sequence of prepro-2S albumin, was found in the purified oil bodies, and mature sesame 2S albumin apparently processed into two subunits (9 and 4 kDa) linked by disulfide bonds was detected in extracts of transgenic seeds. Immunogold labeling revealed that the sesame oleosin and 2S albumin were separately located in oil bodies and protein bodies of embryo cells of transgenic rice seeds. While sesame 2S albumin was also detected in protein bodies of endosperm cells of transgenic seeds, the co-expressed sesame oleosin, probably degraded due to the lack of oil bodies in this tissue, and was not detected. The results provide a new technique for introducing two recombinant polypeptides separately into rice oil bodies and protein bodies from one expression construct.     

Sesame oleosin and prepro-2S albumin expressed as a fusion polypeptide in transgenic rice were split,processed and separately assembled into oil bodies and protein bodies

A recombinant polypeptide containing the precursor protein of a sesame storage protein, 2S albumin, fused to the C-terminus of a sesame oleosin was expressed in transgenic rice seeds under the control of a rice glutelin promoter. The recombinant polypeptide of 32 kDa, equivalent to the resultant molecular mass of sesame oleosin (15 kDa) and prepro-2S albumin (17 kDa), was detected in the endoplasmic reticulum fraction of maturing transgenic rice seeds, but not in the purified oil bodies or the soluble extract of transgenic seeds. However, sesame oleosin presumably fused with a 2 kDa C-terminal appendix originating from the signal sequence of prepro-2S albumin, was found in the purified oil bodies, and mature sesame 2S albumin apparently processed into two subunits (9 and 4 kDa) linked by disulfide bonds was detected in extracts of transgenic seeds. Immunogold labeling revealed that the sesame oleosin and 2S albumin were separately located in oil bodies and protein bodies of embryo cells of transgenic rice seeds. While sesame 2S albumin was also detected in protein bodies of endosperm cells of transgenic seeds, the co-expressed sesame oleosin, probably degraded due to the lack of oil bodies in this tissue, and was not detected. The results provide a new technique for introducing two recombinant polypeptides separately into rice oil bodies and protein bodies from one expression construct.     

小麦胚乳传递细胞发育的结构观察

为从细胞学方面了解小麦产量和品质的形成机制,以扬麦5号为材料,利用光镜和透射电镜观察了小麦颖果发育过程中胚乳传递细胞的结构变化,并探讨了胚乳传递细胞的生理功能。结果表明：（1）胚乳传递细胞是胚乳发育过程中最早分化的细胞类型,它们发生在紧邻胚乳腔的胚乳表层,由位于外侧1~2层糊粉层传递细胞和位于内侧1~2层内胚乳传递细胞构成;（2）颖果发育成熟时,内胚乳传递细胞核衰亡,糊粉层传递细胞核依然完整;（3）胚乳传递细胞发育呈明显的极性,且具时空性;（4）糊粉层传递细胞胞质较浓,富含粗面内质网、线粒体、高尔基体和脂质体;质膜皱褶,在局部区域外翻,形成众多的原生质管;（5）内胚乳传递细胞胞质较稀,液泡化程度较高,富含淀粉体;（6）胚乳传递细胞未加厚以及未形成壁内突的壁区域分布有大量的胞间连丝;（7）胚乳传递细胞中线粒体呈极性分布,即质膜附近线粒体的密度较大。根据胚乳传递细胞的结构特点推测,经胚乳传递细胞的养分输送既可通过质外体途径又可通过共质体途径来完成。     

孕穗期施氮对小麦蛋白质组分积累和蛋白质体发育的影响

为了揭示氮素对小麦蛋白质品质的调节效应,以扬麦9号为材料,研究了孕穗期施氮对小麦籽粒蛋白质组分积累及胚乳细胞蛋白质体发育的影响.结果表明:(1)随花后天数的增加,小麦籽粒清蛋白含量呈现由高到低的变化,球蛋白含量先下降,后期又升高;而醇溶蛋白和麦谷蛋白含量基本呈现由低到高的变化;(2)孕穗期施氮能显著提高醇溶蛋白和麦谷蛋白含量,而对清蛋白和球蛋白含量影响较小;(3)小麦胚乳细胞中蛋白质体约在花后12 d出现,花后16 d蛋白质体开始大量合成,其数目迅速增多,体积变大.成熟后期,蛋白质体因胚乳细胞中大小淀粉体的充实被挤成片层结构而填充在淀粉体缝隙中.胚乳边缘细胞中蛋白质体大而多,中部细胞蛋白质体小而少;(4)孕穗期施氮可促进蛋白质体的形成,使其数目增多.     

施氮时期对扬稻6号颖果发育及稻米品质的影响

通过盆栽试验研究了相同施氮量下不同施氮时期（分蘖期和孕穗期）对扬稻6号颖果发育及稻米品质的影响。与对照相比,增施氮肥（尿素）特别是孕穗肥能显著提高稻米精米率、整精米率、蛋白质含量,降低垩白粒率和直链淀粉含量;在分蘖期和孕穗期施氮肥能明显影响颖果发育,提高粒重,而后者的效果更为明显;在颖果发育过程中不同时期增施氮肥能显著降低颖果的总淀粉和直链淀粉含量,但对支链淀粉含量影响较小;不同时期增施氮肥特别是孕穗肥对淀粉体和蛋白质体的发育及结构均有显著影响,能显著改变籽粒中不同部位特别是腹部淀粉体和蛋白质的分布、数目和形状。与对照相比,淀粉体和蛋白质体的排列更紧密、相互间空隙较少、数量增加、密度增大,淀粉体形状多数呈晶状体。     

水稻应用拿敌稳减氮防病抗倒伏示范效果

通过试验示范探索拿敌稳对水稻穗期氮肥使用量的调控,综合分析结果:水稻施用拿敌稳免去穗肥(氮素)对水稻产量性状影响不大,而且防治稻瘟病效果较好,水稻抗倒伏能力强.建议在水稻始穗期和齐穗期各喷一次药,每次用药量为15 g/667 m2,这样既减少化肥使用量,同时又达到了防治稻瘟病效果.     

Effects of Nitrogen Fertilizer Level on Chlorophyll Fluorescence Characteristics in Flag Leaf of Super Hybrid Rice at Late Growth Stage

To compare the effects of slow-release nitrogen fertilizer at six different levels on the flag leaf chlorophyll fluorescence characteristics of super hybrid rice,a field fertilization experiment was conducted with super hybrid rice Y Liangyou 1 as a test material.The photosynthetic electron transport rate (ETR),effective quantum yield (EQY),photochemical quenching coefficient (q P),and non-photochemical quenching coefficient (NPQ) of flag leaves were measured at the initial heading,full heading,10 d after full heading and 20 d after full heading stages.Results showed that the values of ETR,EQY and q P increased with rice development from initial heading to 20 d after full heading,whereas the NPQ decreased.During the measured stages,ETR,EQY and q P increased initially and then decreased as nitrogen application amount increased,but they peaked at different nitrogen fertilizer levels.The maximum ETR and EQY values appeared at the treatment of 135 kg/hm 2 N.In conclusion,the optimum nitrogen amount for chlorophyll fluorescence characteristics of super hybrid rice was 135 180 kg/hm 2.     

四个不同粒重水稻品种颖果发育的比较

 以粒重差异较大的4个水稻品种为供试材料,采用树脂切片、酶解胚乳细胞和显微观察等方法,比较研究了品种间在颖果生长、胚乳细胞增殖、果皮和胚乳结构等方面的差异,探讨了影响颖果生长的因素。 大粒品种颖果发育时间较小粒品种长,其胚乳细胞数、胚乳干质量及单个胚乳细胞平均干质量均高于小粒品种。在粒重相近的情况下,籼稻颖果发育和淀粉积累快于粳稻。与小粒品种相比,大粒品种子房壁细胞中淀粉粒多,子房壁细胞生长的持续时间长,果皮及背部维管束衰亡迟。 小粒品种胚乳外层细胞在花后7 d已转化成糊粉层细胞,大粒品种胚乳外层细胞要在花后10 d才转化成糊粉层细胞。 大粒品种的库容大和生理活性期长是其颖果能显著增大的生理原因。     

Close Association between Aleurone Traits and Lipid Contents of Rice Grains Observed in Widely Different GeneticResources of Oryza sativa

Abstract

We investigated 321 varieties to identify the aleurone traits that are closely related to the lipid content of rice. Brown rice seeds were cut crosswise near the center with a razor blade and the cut surface was stained with Oil Red O, and then observed under integrated fluorescence microscope (BZ9000). We found wide variations among varieties in the area stained, but the japonica group contained many varieties with a large stained area. We selected 17 representative varieties covering the whole range of stained areas and confirmed the thickness of aleurone layer using Kawamoto’s film method (micro-thin sections that were created with cryomicrotome). Aleurone traits were examined under a light microscope and measured. We found a strong correlation between the area of the aleurone layer and the stained area (r = 0.799***) and between the thickness of the aleurone layer and the thickness of the stained region (r = 0.543*). The area of the stained region therefore provides a fast and effective indicator for selecting varieties for the thickness of the aleurone layer. We also measured the amount (mg per 100 seeds) and proportion of triacylglycerols (TAGs) to 1 g of seed (mg g-¹) in brown rice seeds without embryos. The amount and proportion of TAG to 1 g of seed were both significantly correlated with the area, average thickness and percentage of aleurone layer. This suggests that the aleurone traits will be good indicators for the selection of varieties with high levels of TAG.     

Effects of Nitrogen Application Level on Rice Nutrient Uptake and Ammonia Volatilization

The effects of different nitrogen application levels on nutrient uptake and ammonia volatilization were studied with the rice cultivar Zheyou 12 as a material.The accumulative amounts of nitrogen,phosphorus and potassium in rice plants across all growth stages showed a trend to increase with increasing nitrogen application levels from 0 to 270 kg/hm 2,but decreased at nitrogen application levels exceeding 270 kg/hm 2.Moreover,the accumulative uptake of nitrogen,phosphorus and potassium by the rice plants was increased by application of organic manure in combination with 150 kg/hm 2 nitrogen.The nitrogen uptake was high during the jointing to heading stages.Correlation analysis showed that rice yield was positively correlated with the accumulative uptake of nitrogen,phosphorus and potassium by the rice plants.The highest correlation coefficient observed was between the amount of nitrogen uptake and rice yield.The rate and accumulative amounts of ammonia volatilization increased with increasing nitrogen fertilizer application level.Compared with other stages,the rate and accumulative amount of ammonia volatilization were higher after base fertilizer application.The ammonia volatilization rates in response to the nitrogen application levels of 270 kg/hm 2 and 330 kg/hm 2 were much higher than those in the other treatments.The loss of nitrogen through ammonia volatilization accounted for 23.9% of the total applied nitrogen at the nitrogen application level of 330 kg/hm 2.     

Feasibility study of a tissue-specific approach to barley proteome analysis: aleurone layer,endosperm, embryo and single seeds

Proteome analysis in the pI 4–7 range of aqueous extracts from whole barley seeds results in complex 2D-gel spot patterns that represent proteins from all seed tissues. Proteins were extracted from dissected aleurone layer, embryo and endosperm for separate analysis of these components and comparison with single seed extracts. The analysis showed that while the starchy endosperm comprises approximately 85% of the mature seed dry weight, it contributes less than 50% of the soluble protein to seed extracts. Although the aleurone layer and embryo are minor components of the seed in terms of dry weight, their proteins contribute significantly to the 2D-gel pattern. Distinct but overlapping patterns were observed for the dissected tissues. While whole seed gels contained about 850 spots, endosperm, aleurone layer and embryo gels had about 575, 850 and 1000 spots, respectively, representing a total increase of at least 15%. Protein patterns were also reduced in complexity and this enrichment of subsets of proteins facilitated identification by mass spectrometry of proteins of interest in mature seed tissues and developing endosperm. These techniques can therefore be used with advantage to describe the proteomes of dissected seed tissues.     

孕穗期施氮对小麦胚乳发育的影响

为了从胚乳发育角度研究施氮对小麦品质的影响,以扬麦9号为材料,利用扫描和透射电子显微镜等研究了孕穗期施氮对小麦胚乳细胞游离核分裂周期、细胞数目的增殖、细胞体积的扩大、糊粉层结构及淀粉体和蛋白质体发育的影响.结果表明:(1)胚乳细胞增殖呈"S"型曲线变化,增施孕穗氮肥有利于增加胚乳细胞数目,提高粒重;(2)孕穗期施氮能显著缩短胚乳游离核的分裂周期,促进细胞长和宽的扩大.花后18 d小麦胚乳细胞分裂基本停止,其数目不再增加,以后主要进行细胞体积的扩大;(3)孕穗期施氮可使胚乳糊粉层细胞质变浓,细胞壁增厚,染色性增强;(4)孕穗期施氮可促进胚乳细胞中大淀粉体的形成,有利于大淀粉体和蛋白质体数目增多,蛋白质被染色更浓;(5)孕穗期施氮可减少最终籽粒小淀粉体的数目,提高淀粉体和蛋白质体的充实程度.     

Localization of iron in rice grain using synchrotron X-ray fluorescence microscopy and high resolution secondary ion mass spectrometry

Cereal crops accumulate low levels of iron (Fe) of which only a small fraction (5–10%) is bioavailable in human diets. Extensive co-localization of Fe in outer grain tissues with phytic acid, a strong chelator of metal ions, results in the formation of insoluble complexes that cannot be digested by humans. Here we describe the use of synchrotron X-ray fluorescence microscopy (XFM) and high resolution secondary ion mass spectrometry (NanoSIMS) to map the distribution of Fe, zinc (Zn), phosphorus (P) and other elements in the aleurone and subaleurone layers of mature grain from wild-type and an Fe-enriched line of rice (Oryza sativa L.). The results obtained from both XFM and NanoSIMS indicated that most Fe was co-localized with P (indicative of phytic acid) in the aleurone layer but that a small amount of Fe, often present as “hotspots”, extended further into the subaleurone and outer endosperm in a pattern that was not co-localized with P. We hypothesize that Fe in subaleurone and outer endosperm layers of rice grain could be bound to low molecular weight chelators such as nicotianamine and/or deoxymugineic acid.