New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana

Background and Aims

The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.

Methods

In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.

Key Results

Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.

Conclusions

This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.     

基因型与培养条件对羽衣甘蓝小孢子胚胎发生的影响

本文详细地研究了小孢子发育时期、基因型与培养条件对羽衣甘蓝小孢子胚胎发生的影响,建立了一个稳定、高频地获得小孢子胚胎的有效体系。结果表明,不同基因型材料相同大小的花蕾其小孢子发育时期存在很大差异,需针对不同基因型材料选取适合大小的花蕾。供试的37个基因型中,有20个获得了胚状体,占供试材料的54．1％,其中基因型‘桃舞’获得了最高的出胚率,为123．6个·皿-1。自交系的出胚率比商业品种和F,代杂种的出胚率要低得多,且自交代数越高,小孢子的胚胎发生能力就越弱。在热激培养48hN加液培养对小孢子的发育能起到积极作用,向培养基中添加激素和活性炭对小孢子的胚胎发生无促进作用。     

Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning

The inaccessibility of the zygote and proembryos of angiospermswithin the surrounding maternal and filial tissues has hamperedstudies on early plant embryogenesis. Somatic and gametophyticembryo cultures are often used as alternative systems for molecularand biochemical studies on early embryogenesis, but are notwidely used in developmental studies due to differences in theearly cell division patterns with seed embryos. A new Brassicanapus microspore embryo culture system, wherein embryogenesishighly mimics zygotic embryo development, is reported here.In this new system, the donor microspore first divides transverselyto form a filamentous structure, from which the distal cellforms the embryo proper, while the lower part resembles thesuspensor. In conventional microspore embryogenesis, the microsporedivides randomly to form an embryonic mass that after a whileestablishes a protoderm and subsequently shows delayed histodifferentiation.In contrast, the embryo proper of filament-bearing microspore-derivedembryos undergoes the same ordered pattern of cell divisionand early histodifferentiation as in the zygotic embryo. Thisobservation suggests an important role for the suspensor inearly zygotic embryo patterning and histodifferentiation. Thisis the first in vitro system wherein single differentiated cellsin culture can efficiently regenerate embryos that are morphologicallycomparable to zygotic embryos. The system provides a powerfulin vitro tool for studying the diverse developmental processesthat take place during the early stages of plant embryogenesis. Key words: Brassica napus, microspore embryogenesis, pattern formation, polarity, suspensor, zygotic embryogenesis     

An overview of preliminary studies on the development of doubled haploid protocols for nutraceutical species

There is very little activity underway to improve the genetics of herbs, spices, and nutraceutical crops. Much of the industry relies on the harvest of “wild” plants; therefore, the potential for variability in performance and active ingredients is high. This presents significant challenges for an industry that is striving to achieve market credibility and meet current regulatory standards. Uniform varieties would also be beneficial for use in clinical trials. The development of plants performing consistently in cultivation under various environmental conditions and producing a stable quality and quantity of desired active ingredients cannot solely rely on traditional plant breeding, but must be supported by the development of tissue culture methods targeted to the species of interest. We have screened over 80 herb, spice, and nutraceutical species for microspore culture response using the Brassica napus microspore culture protocol. The majority of the species did not respond. Swelling and initial divisions of the microspores were observed in some species. Embryogenesis, however, was observed in the Apiaceae and the Caryophyllaceae. Species within these families were selected for further optimization. Improvements in embryogenic frequency were observed in both families. Haploid and doubled haploid plants have been regenerated in anise (Pimpinella anisum), carrot (Daucus carota), caraway (Carum carvi), dill (Anethum graveolens), fennel (Foeniculum vulgare), lovage (Levisticum officinale), laceflower (Amni majus), parsnip (Petroselinum crispum), and cow cockle (Saponaria vaccaria).     

ABNORMAL POLLEN VACUOLATION1 (APV1) is required for male fertility by contributing to anther cuticle and pollen exine formation in maize

Anther cuticle and pollen exine are the major protective barriers against various stresses. The proper functioning of genes expressed in the tapetum is vital for the development of pollen exine and anther cuticle. In this study, we report a tapetum‐specific gene, Abnormal Pollen Vacuolation1 (APV1), in maize that affects anther cuticle and pollen exine formation. The apv1 mutant was completely male sterile. Its microspores were swollen, less vacuolated, with a flat and empty anther locule. In the mutant, the anther epidermal surface was smooth, shiny, and plate‐shaped compared with the three‐dimensional crowded ridges and randomly formed wax crystals on the epidermal surface of the wild‐type. The wild‐type mature pollen had elaborate exine patterning, whereas the apv1 pollen surface was smooth. Only a few unevenly distributed Ubisch bodies were formed on the apv1 mutant, leading to a more apparent inner surface. A significant reduction in the cutin monomers was observed in the mutant. APV1 encodes a member of the P450 subfamily, CYP703A2‐Zm, which contains 530 amino acids. APV1 appeared to be widely expressed in the tapetum at the vacuolation stage, and its protein signal co‐localized with the endoplasmic reticulum (ER) signal. RNA‐Seq data revealed that most of the genes in the fatty acid metabolism pathway were differentially expressed in the apv1 mutant. Altogether, we suggest that APV1 functions in the fatty acid hydroxylation pathway which is involved in forming sporopollenin precursors and cutin monomers that are essential for the development of pollen exine and anther cuticle in maize.     

<i>OsMAPK6</i> Affects Male Fertility by Reducing Microspore Number and Delaying Tapetum Degradation in <i>Oryza sativa</i> L.

The mitogen-activated protein kinase (MAPK) cascade is important in stress signal transduction and plant development. In the present study, we identified a rice (Oryza sativa L.) mutant with reduced fertility, Oryza sativa mitogen-activated protein kinase 6 (osmapk6), which harbored a mutated MAPK gene. Scanning and transmission electron microscopy, quantitative RT-PCR analysis, TUNEL assays, RNA in situ hybridization, longitudinal and transverse histological sectioning, and map-based cloning were performed to characterize the osmapk6 mutant. The gene OsMAPK6 was expressed throughout the plant but predominantly in the microspore mother cells, tapetal cells, and microspores in the anther sac. Compared with the wild type, the total number of microspores was reduced in the osmapk6 mutant. The formation of microspore mother cells was reduced in the osmapk6 anther sac at an early stage of anther development, which was the primary reason for the decrease in the total number of microspores. Programmed cell death of some tapetal cells was delayed in osmapk6 anthers and affected exine formation in neighboring microspores. These results suggest that OsMAPK6 plays pivotal roles in microspore mother cell formation and tapetal cell degradation.     

同一居群韭莲不同植株减数分裂行为差异的遗传分析

对韭莲(2n=48)小孢子母细胞减数分裂及小孢子发育进行研究。结果显示同一居群植株的减数分裂行为存在明显差异。多数韭莲植株小孢子母细胞减数分裂存在少量落后染色体、微核等现象,平均每株中具有异常分离行为的母细胞占14.02%,小孢子发育正常,但花粉无活力。并首次从减数分裂后期Ⅰ的特殊的细胞学形态证明韭莲是臂内倒位杂合体。而少数植株韭莲的小孢子母细胞减数分裂极其紊乱,后期Ⅰ出现多极分离、大量落后染色体,小孢子母细胞减数分裂总异常分离高达94.3%。四分孢子期多分孢子体高达73.4%。分析认为:前者减数分裂行为异常的原因主要由染色体结构变异所致,而后者的原因除染色体结构变异外,还可能与控制纺锤体形成的基因突变有关。     

黄瓜花器形态发生、小孢子发育与花药培养

孢子发育时期与花蕾形态特征、花药颜色具有相关性。本试验目的是确定黄瓜花药的最佳培养时期,并确定对应的选蕾标准。对基因型、预处理等花药培养主要影响因素研究结果表明：不同基因型小孢子单核中后期花蕾长度不同,形态上没有差异;同一基因型的小孢子发育不同时期的花器特征和诱导率均有差异,单核中后期花药诱导率最高;低温预处理有利于保持小孢子的活力;花药培养中以黑暗中4℃、48～72h低温预处理最有助于愈伤发生。实验结果表明,黄瓜花药培养中小孢子最佳培养时期为单核中后期,取蕾标准为：花蕾长度0．90～1．50cm,绿色,瓣尖未张开,花药白绿色或淡绿色。     

Plant regeneration from isolated microspores of Triticum aestivum

Summary Wheat microspores were isolated, without prior anther culture, from a range of genotypes and cultured to regenerate self-fertile plants. Microspores were isolated using a microblender and competent microspores were enriched by gradient centrifugation. The use of maltose as the sole carbohydrate in the culture medium and co-culture of microspores with wheat or barley ovaries were critical for development of microspore-derived embryos. Results were also improved when spikes were pretreated by emersion of the basal ends of detached heads in water at 25°C for 2d. This procedure leads to highly reproducible production of plants.