Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H₂DCFDA‐staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into ‘low‐ROS’ and ‘high‐ROS’ subpopulations. Pollen germination assays following fluorescence‐activated cell sorting revealed that the high‐ROS pollen germinated with a frequency that was 35‐fold higher than the low‐ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose‐dependent alterations in DCF‐fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry‐based approaches to investigate metabolic changes during stress responses in pollen.     

Morphological and functional seed traits of the wild medicinal plant Dioscorea strydomiana,the most threatened yam in the world

• Morphological and functional seed traits have important roles in characterising the species regeneration niche and help to understand the reproductive biology of rare and threatened plants, which can thus support appropriate plant conservation measures.
• Seed morphometric and dispersal kinetics of the critically endangered Dioscorea strydomiana were measured and compared with those of four other Dioscorea species, and seed germination response under constant temperatures (5–35 °C) was compared with that of the congeneric and widespread D. sylvatica.
• Seed mass of D. strydomiana (ca. 14 mg) was twice that of D. sylvatica, but similar to or smaller than the other species examined. Seeds of D. strydomiana have the lowest speed of descent and lowest variability in most of the morphological traits considered, suggesting lower phenotypic plasticity but higher variance in the wing‐loading value. Seeds of D. strydomiana reached maximum germination at 15 °C (ca. 47%), which decreased slightly to ca. 37% at 25 °C and was completely inhibited at 35 °C. D. sylvatica seeds started to germinate at 10 °C (ca. 3%), reached 75–80% germination at 15–20 °C and maximum (ca. 90%) at 25–30 °C. Base temperatures for germination (T_b) were 9.3 and 5.7 °C, for D. strydomiana and D. sylvatica, respectively. Due to the higher germination percentages of D. sylvatica, ceiling and optimum temperatures could also be modelled for this species, suggesting higher sensitivity to high temperature for seeds of D. strydomiana.
• The detected poor seed lot quality of D. strydomiana suggests difficulties in reproduction from seed, highlighting the need for further investigation and conservation actions for this threatened yam species.

     

Investigating seed dormancy in cotton (Gossypium hirsutum L.): understanding the physiological changes in embryo during after‐ripening and germination

• The dormancy of seeds of upland cotton can be broken during dry after‐ripening, but the mechanism of its dormancy release remains unclear.
• Freshly harvested cotton seeds were subjected to after‐ripening for 180 days. Cotton seeds from different days of after‐ripening (DAR) were sampled for dynamic physiological determination and germination tests. The intact seeds and isolated embryos were germinated to assess effects of the seed coat on embryo germination. Content of H₂O₂ and phytohormones and activities of antioxidant enzymes and glucose‐6‐phosphate dehydrogenase were measured during after‐ripening and germination.
• Germination of intact seeds increased from 7% upon harvest to 96% at 30 DAR, while embryo germination improved from an initial rate of 82% to 100% after 14 DAR. Based on T₅₀ (time when 50% of seeds germinate) and germination index, the intact seed and isolated embryo needed 30 and 21 DAR, respectively, to acquire relatively stable germination. The content of H₂O₂ increased during after‐ripening and continued to increase within the first few hours of imbibition, along with a decrease in abscisic acid (ABA) content. A noticeable increase was observed in gibberellic acid content during germination when ABA content decreased to a lower level. Coat removal treatment accelerated embryo absorption of water, which further improved the accumulation of H₂O₂ and changed peroxidase content during germination.
• For cotton seed, the alleviation of coat‐imposed dormancy required 30 days of after‐ripening, accompanied by rapid dormancy release (within 21 DAR) in naked embryos. H₂O₂ acted as a core link between the response to environmental changes and induction of other physiological changes for breaking seed dormancy.

     

叶面喷施PP_(333)和6-BA对甜荞花芽分化及内源激素的影响

以甜荞品种‘北早生’和‘赤甜荞1号’为材料,利用多效唑(PP_(333))和6-苄基腺嘌呤(6-BA)在不同幼苗时期进行叶片喷施处理,并采用扫描电镜观察花芽分化过程,记录开花时间,调查开花和结实数,通过液质联用方法检测3种内源激素生长素(IAA)、赤霉素(GA_3)、脱落酸(ABA)及人工合成细胞分裂素(6-BA)含量,探究两种植物生长调节剂对甜荞的花芽分化及内源激素含量的影响,为甜荞花期调控提供依据。结果显示:(1)第二片真叶期喷施100mg·L~(-1)PP_(333)和150mg·L~(-1)6-BA能显著提高甜荞结实率和单株产量,两种处理的结实率分别较对照提高49.5%、39.4%,单株产量分别提高41.8%、23.0%。(2)观察甜荞花芽分化过程并将其划分5个时期,分别为生长锥分化前期、生长锥分化期、小花原基分化期、雌雄蕊分化期和雌雄蕊形成期,在第二片真叶期喷施PP_(333)和6-BA均加快了甜荞花芽分化进程,使其开花时间提前。(3)喷施PP_(333)在幼苗期和现蕾期降低了内源IAA、6-BA含量,增加GA_3、ABA含量,而在盛花期增加内源IAA、6-BA含量,降低ABA、GA_3含量;喷施6-BA在幼苗期和现蕾期增加了内源GA_3、IAA含量,在盛花期降低了内源GA_3、IAA含量,3个时期均降低了内源ABA含量,增加了6-BA含量。研究发现,第二片真叶期是调控甜荞小花发育的关键时期,在此时叶面喷施100mg·L~(-1)PP_(333)和150mg·L~(-1)6-BA的调控效果最佳,可加快甜荞花芽分化进程,使开花时间提前,增加单株结实粒数,提高甜荞品种的结实率和单株产量;PP_(333)主要是通过减少甜荞开花数,增强弱势小花活力来提高结实粒数,而6-BA主要是利用增加开花数,提高有效可孕小花数来增加结实粒数。     

濒危植物中甸刺玫种子休眠及其生态学意义

对中甸刺玫Rosa praelucens种子结构及其透水性,赤霉素处理对胚的影响,果壳、种皮、胚乳的粗提物活性进行研究。结果表明：(1) 中甸刺玫外种皮是由多层排列紧密的厚壁细胞组成,内种皮为坚硬致密的栅栏组织;(2) 种皮对种子的吸胀阻碍较大, 未处理的种子吸水率较低,吸水13 d后增加量为18.82%;(3) 果实结籽率为0.69%,多数果实中没有饱满的种子;(4) 赤霉素100 mg·kg-1预处理种子可加快胚的萌发速率;(5) 胚乳、种皮、果壳中存在内源抑制物。中甸刺玫种子的休眠是由其形态和生理特点引起的综合休眠。     

莼菜休眠芽解除休眠及生根的研究

莼菜为世界著名珍稀水生蔬菜,具有很高的食用和药用价值。已有研究显示,可通过冬芽实现莼菜复壮,找出休眠解除的最佳方法是复壮的基本前提。本实验以莼菜冬季型休眠芽（冬芽）为实验材料,通过光照、温度等处理探求莼菜冬芽的休眠解除方法。莼菜冬芽生根率随着光照强度的增加,其生根率由全黑暗0%显著增长至（16.0±1.3）%;温度和处理时间对莼菜冬芽生根率、根长、生根根数和含水量有显著性影响。其中,35 °C水浴处理6 h后各项指标均到达较大值,上述指标分别为（96.1±2.8）%、（7.2±0.2） cm、74.5±6.0和（83.6±0.3）%;40 °C（6 h）、45 °C（1、3和6 h）和50 °C（10、20和30 min）水浴处理后莼菜冬芽绝大部分失绿变黄;随着培养时间的延长,其冬芽全部腐烂。光照能够促进莼菜冬芽生根;土培生根效果较为显著;适度水浴加热处理能有效解除莼菜冬芽的休眠,35 °C水浴处理6 h为最佳处理方案。     

A cellular lineage analysis of the chick limb bud

The chick limb bud has been used as a model system for studying pattern formation and tissue development for more than 50 years. However, the lineal relationships among the different cell types and the migrational boundaries of individual cells within the limb mesenchyme have not been explored. We have used a retroviral lineage analysis system to track the fate of single limb bud mesenchymal cells at different times in early limb development. We find that progenitor cells labeled at stage 19-22 can give rise to multiple cell types including clones containing cells of all five of the major lateral plate mesoderm-derived tissues (cartilage, perichondrium, tendon, muscle connective tissue, and dermis). There is a bias, however, such that clones are more likely to contain the cell types of spatially adjacent tissues such as cartilage/perichondrium and tendon/muscle connective tissue. It has been recently proposed that distinct proximodistal segments are established early in limb development; however our analysis suggests that there is not a strict barrier to cellular migration along the proximodistal axis in the early stage 19-22 limb buds. Finally, our data indicate the presence of a dorsal/ventral boundary established by stage 16 that is inhibitory to cellular mixing. This boundary is demarcated by the expression of the LIM-homeodomain factor lmx1b.     

A highly conserved Wnt-dependent TCF4 binding site within the proximal enhancer of the anti-myogenic Msx1 gene supports expression within Pax3-expressing limb bud muscle precursor cells

The product of the Msx1 gene is a potent inhibitor of muscle differentiation. Msx1 is expressed in muscle precursor cells of the limb bud that also express Pax3. It is thought that Msx1 may facilitate distal migration by delaying myogenesis in these cells. Despite the role played by Msx1 in inhibiting muscle differentiation, nothing is known of the mechanisms that support the expression of the Msx1 gene within limb bud muscle precursor cells. In the present study we have used a combination of comparative genomics, mouse transgenic analysis, in situ hybridisation and immunohistochemistry to identify a highly conserved and tissue-specific regulatory sub-domain within the previously characterised Msx1 gene proximal enhancer element that supports the expression of the Msx1 gene in Pax3-expressing mouse limb pre-muscle masses. Furthermore, using a combination of in situ hybridisation, in vivo ChIP assay and transgenic explant culture analysis we provide evidence that Msx1 expression in limb bud muscle precursor cells is dependent on the canonical Wnt/TCF signalling pathway that is important in muscle shape formation. The results of these studies provide evidence of a mechanistic link between the Wnt/TCF and the Msx1/Pax3/MyoD pathways within limb bud muscle precursor cells.     

‘赛蜜酥1号’枣及其芽变品系果实的性状差异北大核心CSCD

该试验以‘赛蜜酥1号’枣及其芽变品系‘赛蜜酥2号’果实为试验材料,运用流式细胞术对它们的倍性进行鉴定,采用石蜡切片法进行果实细胞组学的观察,并对两者果实生长发育过程中的内外观品质及决定果实口感的蔗糖代谢相关酶活性进行比较分析,为深入研究芽变对枣果实口感、品质造成的影响提供理论支持。结果表明:(1)‘赛蜜酥2号’芽变品系的细胞倍性未发生改变,仍为二倍体。(2)两个枣品种(系)果实在外部形态上有明显区别,‘赛蜜酥1号’为卵圆型,‘赛蜜酥2号’为扁圆型,且后者的果形指数成熟时大于1,单果质量高于前者,核小、可食率更高,皮薄果实更加酥脆;‘赛蜜酥1号’果皮的蜡质层厚度一直大于其芽变枣,两者角质层厚度和表皮层厚度的变化趋势基本一致,但厚度之间具有显著差异。(3)影响果实细胞生长、分裂,果实脱落的各类激素水平在品种间均存在显著差异,‘赛蜜酥2号’IAA和GA_(3)含量显著高于‘赛蜜酥1号’,使之果形更大。(4)两个枣品种(系)果实可溶性糖、可滴定酸、维生素C、植物淀粉、可溶性蛋白含量等随发育时期的变化趋势一致,但含量水平之间存在差异,‘赛蜜酥2号’果实的糖含量、维生素C含量更高,可滴定酸含量更低,口感更甘甜。(5)两品种枣果实的蔗糖代谢相关酶活性在果实迅速生长的膨大期都存在着显著差异,也导致‘赛蜜酥2号’果实甜度明显高于‘赛蜜酥1号’。研究发现,两个枣品种均为二倍体,它们在果实外部形态上存在明显区别,易于分辨;芽变品系‘赛蜜酥2号’果实更大,核小、可食率更高,皮薄更加酥脆,口感更甘甜。