Identification and Genetic Analysis of a Novel Allelic Variation of <i>Brittle-1</i> 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.     

Multi‐scale spatial heterogeneity of pectic rhamnogalacturonan I (RG–I) structural features in tobacco seed endosperm cell walls

Plant cell walls are complex configurations of polysaccharides that fulfil a diversity of roles during plant growth and development. They also provide sets of biomaterials that are widely exploited in food, fibre and fuel applications. The pectic polysaccharides, which comprise approximately a third of primary cell walls, form complex supramolecular structures with distinct glycan domains. Rhamnogalacturonan I (RG–I) is a highly structurally heterogeneous branched glycan domain within the pectic supramolecule that contains rhamnogalacturonan, arabinan and galactan as structural elements. Heterogeneous RG–I polymers are implicated in generating the mechanical properties of cell walls during cell development and plant growth, but are poorly understood in architectural, biochemical and functional terms. Using specific monoclonal antibodies to the three major RG–I structural elements (arabinan, galactan and the rhamnogalacturonan backbone) for in situ analyses and chromatographic detection analyses, the relative occurrences of RG–I structures were studied within a single tissue: the tobacco seed endosperm. The analyses indicate that the features of the RG–I polymer display spatial heterogeneity at the level of the tissue and the level of single cell walls, and also heterogeneity at the biochemical level. This work has implications for understanding RG–I glycan complexity in the context of cell‐wall architectures and in relation to cell‐wall functions in cell and tissue development.     

Rice endosperm is cost‐effective for the production of recombinant griffithsin with potent activity against HIV

Protein microbicides containing neutralizing antibodies and antiviral lectins may help to reduce the rate of infection with human immunodeficiency virus (HIV) if it is possible to manufacture the components in large quantities at a cost affordable in HIV‐endemic regions such as sub‐Saharan Africa. We expressed the antiviral lectin griffithsin (GRFT), which shows potent neutralizing activity against HIV, in the endosperm of transgenic rice plants (Oryza sativa), to determine whether rice can be used to produce inexpensive GRFT as a microbicide ingredient. The yield of ^OSGRFT in the best‐performing plants was 223 μg/g dry seed weight. We also established a one‐step purification protocol, achieving a recovery of 74% and a purity of 80%, which potentially could be developed into a larger‐scale process to facilitate inexpensive downstream processing. ^OSGRFT bound to HIV glycans with similar efficiency to GRFT produced in Escherichia coli. Whole‐cell assays using purified ^OSGRFT and infectivity assays using crude extracts of transgenic rice endosperm confirmed that both crude and pure ^OSGRFT showed potent activity against HIV and the crude extracts were not toxic towards human cell lines, suggesting they could be administered as a microbicide with only minimal processing. A freedom‐to‐operate analysis confirmed that GRFT produced in rice is suitable for commercial development, and an economic evaluation suggested that 1.8 kg/ha of pure GRFT could be produced from rice seeds. Our data therefore indicate that rice could be developed as an inexpensive production platform for GRFT as a microbicide component.     

侧金盏胚和胚乳发育的细胞胚胎学研究

利用石蜡切片技术对毛茛科植物侧金盏胚及胚乳发育进行了研究,以明确其胚胎发育的特征,为毛茛科植物的系统研究提供资料。结果表明:(1)侧金盏胚的发育属于柳叶菜型,胚乳发育为核型;初生胚乳核的分裂早于合子的第一次分裂。(2)种子成熟时,种胚尚未分化完全,尚处于球形胚后期或心形胚早期阶段,整个胚发育大约需要50~60d。(3)侧金盏种子存在明显的形态生理休眠现象,经后熟作用逐渐完成种胚的分化与生长,形成子叶形胚;侧金盏种子在相同处理条件下胚分化和发育的速度存在差异。     

Nuclear fusions contribute to polyploidization of the gigantic nuclei in the chalazal endosperm of<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Somatic polyploidization is recognized as a means to increase gene expression levels in highly active metabolic cells. The most common mechanisms are endoreplication, endomitosis and cell fusion. In animals and plants the nuclei of multinucleate cells are usually prevented from fusing. Here, we report that the nuclei from the syncytial cyst of the chalazal endosperm of Arabidopsis thaliana (L.) Heynh. are polyploid with some intermediate ploidy levels that cannot be attributed to endoreplication, suggesting nuclear fusion. Analysis of isolated nuclei, together with fluorescent in situ hybridization (FISH), revealed that nuclei from the chalazal endosperm are two or three times bigger than the nuclei from the peripheral endosperm and have a corresponding increase in ploidy. Together with the consistent observation of adjoined nuclei, we propose that nuclear fusion contributes, at least in part, to the process of polyploidization in the chalazal endosperm. Confocal analysis of intact seeds further suggested that free nuclei from the peripheral endosperm get incorporated into the chalazal cyst and likely participate in nuclear fusions.Abbreviations BAC Bacterial artificial chromosome - CZE Chalazal endosperm - DAPI 4,6-Diamino-2-phenylindole - FISH Fluorescent in situ hybridization - NOR Nucleolar organizing region - NCD Nuclear cytoplasmic domain - PEN Peripheral endosperm     

Neuronal and muscular alterations caused by two wheat endosperm proteins,puroindoline-a and alpha1-purothionin,are due to ion pore formation

Using the patch-clamp technique it was found that the toxicity of the two wheat endosperm proteins puroindoline-a and alpha1-purothionin probably results from the dissipation of ion concentration gradients essential for the maintenance of cellular homeostasis.Abbreviations PIN-a puroindoline-a - PTH alpha1-purothionin Presented at the Biophysical Society Meeting on Ion Channels—from structure to disease held in May 2003, Rennes, France     

Fertilization in Arabidopsis thaliana wild type: developmental stages and time course

We describe some previously uncharacterised stages of fertilization in Arabidopsis thaliana and provide for the first time a precise time course of the fertilization process. We hand-pollinated wild type pistils with wild type pollen (Columbia ecotype), fixed them at various times after pollination, and analysed 600 embryo sacs using Confocal Laser Scanning Microscopy. Degeneration of one of the synergid cells starts at 5 Hours After Pollination (HAP). Polarity of the egg changes rapidly after this synergid degeneration. Karyogamy is then detected by the presence of two nucleoli of different diameters in both the egg and central cell nuclei, 7-8 HAP. Within the next hour, first nuclear division takes place in the fertilized central cell and two nucleoli can then be seen transiently in each nucleus produced. In a second set of experiments, we hand-pollinated wild type pistils with pollen from a transgenic promLAT52::EGFP line that expresses EGFP in its pollen vegetative cell. Release of the pollen tube contents into the synergid cell could be detected in living material. We show that the timing of synergid degeneration and pollen tube release correlate well, suggesting that either the synergid cell degenerates at the time of pollen tube discharge or very shortly before it. These observations and protocols constitute an important basis for the further phenotypic analysis of mutants affected in fertilization.